Heterodimers of soluble interferon receptors and uses thereof

ABSTRACT

The present disclosure provides soluble interferon receptors. The methods of the disclosure can be used to treat or prevent a condition associated with an abnormal immune response.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.16/109,672, filed on Aug. 22, 2018, pending, which claims the benefit ofU.S. Provisional Application Ser. No. 62/548,737 filed on Aug. 22, 2017.The entire contents of the above-referenced provisional patentapplications are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format via EFS-Web and is herebyincorporated by reference in its entirety. Said ASCII copy, created Feb.4, 2021, is named “SBN-001DV_Sequence_Listing.txt” and is 477515 bytesin size.

BACKGROUND

Systemic lupus erythematosus (SLE) is a chronic, multisystem, autoimmunedisease that is characterized by diverse disease manifestationsimpacting multiple organs, including the skin, CNS, joints, vasculature,and kidneys. There is evidence to suggest that interferon (IFN) isoverproduced in subjects with SLE and contributes to the systemicinflammation that is characteristic of SLE. In particular, Type Iinterferons are the prominent cytokines in SLE and are stronglycorrelated with disease activity and nephritis. Type I interferons are asubgroup of interferon proteins that include IFN-α, INF-β, IFN-ε, -κ,-τ, -ζ, IFN-ω, IFN-ν; all of which bind to the IFN-α receptor (IFNAR)which is comprised of two distinct polypeptide chains, IFNAR1 andIFNAR2. Thus, there exists a need for a means to remove the interferonand/or reduce the inflammation in subjects in need thereof.

SUMMARY OF THE INVENTION

The disclosure relates, in part, to soluble interferon receptors whichare capable of binding interferon (e.g., IFN-α, IFN-β). Such solubleinterferon receptors are useful to inhibit interferon activity. In someaspects, the soluble interferon receptors of the disclosure areheterodimeric constructs. In some aspects, the disclosure relates tosoluble interferon receptors that are beneficial in the treatment ofdiseases characterized by interferon (e.g., SLE, Sjögren's syndrome).

The present disclosure provides heterodimers which binds type Iinterferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an interferon receptor 1(IFNAR1) domain operatively coupled with or without a linker domain to amutant Fc domain, and wherein the second polypeptide comprises aninterferon receptor 2 (IFNAR2) domain operatively coupled with orwithout a linker domain to a mutant Fc domain.

In some aspects, the present disclosure provides a heterodimer whichbinds type I interferons comprising a first polypeptide and a secondpolypeptide, wherein the first polypeptide comprises an IFNAR1 domainoperatively coupled without a linker domain to a mutant Fc domain, andwherein the second polypeptide comprises an IFNAR2 domain operativelycoupled without a linker domain to a mutant Fc domain. In some aspects,the heterodimer of the disclosure in which each of the first and secondpolypeptides is linked directly to a mutant Fc domain (without a linker)has increased binding to type I interferons relative to a heterodimer inwhich each of the first and second polypeptides comprise a polypeptidelinker domain.

In some aspects, the heterodimer of the disclosure comprises a firstpolypeptide and a second polypeptide, wherein the first polypeptidecomprises an IFNAR1 domain operatively coupled with a linker domain(e.g., a polypeptide linker) to a mutant Fc domain, and wherein thesecond polypeptide comprises an IFNAR2 domain operatively coupled with alinker domain (e.g., a polypeptide linker) to a mutant Fc domain. Insome aspects, the heterodimer of the disclosure comprises a firstpolypeptide, wherein the first polypeptide comprises a polypeptidelinker (e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5,1, 2, 3, 4, or 5). In some aspects, the heterodimer of the disclosurecomprises a second polypeptide, wherein the second polypeptide comprisesa polypeptide linker (e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein nis 1-10, 2-5, 1, 2, 3, 4, or 5). In some aspects, the polypeptide linkeris about 1-50, about 5-40, about 10-30, or about 15-20 amino acids inlength. In some aspects, the polypeptide linker is about 20 amino acidsor less, about 15 amino acids or less, about 10 amino acids or less, orabout 5 amino acids or less in length. In some aspects, the polypeptidelinker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length.

In some aspects, the heterodimer of the disclosure binds type Iinterferons selected from interferon-α (INFα), interferon-β (INFβ), orboth INFα and INFβ. In some aspects, the type I interferon is INFα. Insome aspects, the type I interferon is INFβ. In some aspects, the type Iinterferon is both INFα and INFβ. In other aspects, the heterodimer ofthe disclosure inhibits an activity of INFα. In some aspects, theheterodimer of the disclosure inhibits an activity of INFβ. In someaspects, the heterodimer of the disclosure inhibits an activity of bothINFα and INFβ. In some aspects, the heterodimer of the disclosureinhibits induction of type I interferon (IFN) gene expression.

In some aspects, the heterodimer of the disclosure comprises a firstpolypeptide and a second polypeptide, wherein the first polypeptidecomprises an IFNAR1 domain operatively coupled with or without a linkerdomain (e.g., a polypeptide linker) to a mutant Fc domain, and whereinthe second polypeptide comprises an IFNAR2 domain operatively coupledwith or without a linker domain (e.g., a polypeptide linker) to a mutantFc domain. In some aspects, the mutant Fc domain of each of the firstand second polypeptides comprises a mutant human immunoglobulin Fcdomain. In some aspects, the mutant Fc domain of each of the first andsecond polypeptides comprises one or more mutations in a CH2 domain. Insome aspects, the mutant Fc domain of each of the first and secondpolypeptides comprises one or more mutations in a CH3 domain. In someaspects, the mutant Fc domain of each of the first and secondpolypeptides comprises one or more mutations in a CH2 domain and one ormore mutations in a CH3 domain. In some aspects, the mutant Fc domain ofeach of the first and second polypeptides comprises a mutant human IgG1Fc domain. In some aspects, the mutant Fc domain of each of the firstand second polypeptides comprises a mutant human IgG4 domain.

In some aspects, the disclosure provides heterodimers which bind type 1interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with or without a linker domain (e.g., a polypeptide linker,e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2,3, 4, or 5) to a mutant Fc domain comprising a mutation T366Y, andwherein the second polypeptide comprises an IFNAR2 domain operativelycoupled with or without a linker domain (e.g., a polypeptide linker,e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2,3, 4, or 5) to a mutant Fc domain comprising mutation Y407T, accordingto EU numbering. In some aspects, the first and second polypeptide eachcomprise a mutant human IgG1 Fc domain. In some aspects, the first andsecond polypeptide each comprise a mutant human IgG4 Fc domain. In someaspects, the first and second polypeptide each comprise a polypeptidelinker, wherein the polypeptide linker is a Gly/Ser linker, e.g.,(G₄S)_(n), wherein n is 1-10, 2-5, 1, 2, 3, 4, or 5.

In some aspects, the disclosure provides heterodimers which bind type 1interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with or without a linker domain (e.g., a polypeptide linker,e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2,3, 4, or 5) to a mutant Fc domain comprising a mutation Y407T, andwherein the second polypeptide comprises an IFNAR2 domain operativelycoupled with or without a linker domain (e.g., a polypeptide linker,e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2,3, 4, or 5) to a mutant Fc domain comprising mutation T366Y, accordingto EU numbering. In some aspects, the first and second polypeptide eachcomprise a mutant human IgG1 Fc domain. In some aspects, the first andsecond polypeptide each comprise a mutant human IgG4 Fc domain. In someaspects, the first and second polypeptide each comprise a polypeptidelinker, wherein the polypeptide linker is a Gly/Ser linker, e.g.,(G₄S)_(n), wherein n is 1-10, 2-5, 1, 2, 3, 4, or 5.

In some aspects, the disclosure provides heterodimers which bind type 1interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with or without a linker domain (e.g., a polypeptide linker,e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2,3, 4, or 5) to a mutant Fc domain comprising a mutation T366W, andwherein the second polypeptide comprises an IFNAR2 domain operativelycoupled with or without a linker domain (e.g., a polypeptide linker,e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2,3, 4, or 5) to a mutant Fc domain comprising mutations T366S, L368A andY407V, according to EU numbering. In some aspects, the first and secondpolypeptide each comprise a mutant human IgG1 Fc domain. In someaspects, the first and second polypeptide each comprise a polypeptidelinker, wherein the polypeptide linker is a Gly/Ser linker, e.g.,(G₄S)_(n), wherein n is 1-10, 2-5, 1, 2, 3, 4, or 5.

In some aspects, the disclosure provides heterodimers which bind type 1interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with or without a linker domain (e.g., a polypeptide linker,e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2,3, 4, or 5) to a mutant Fc domain comprising mutations T366S, L368A andY407V, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled with or without a linker domain (e.g., a polypeptidelinker, e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5,1, 2, 3, 4, or 5) to a mutant Fc domain comprising a mutation T366W,according to EU numbering. In some aspects, the first and secondpolypeptide each comprise a mutant human IgG1 Fc domain. In someaspects, the first and second polypeptide each comprise a polypeptidelinker, wherein the polypeptide linker is a Gly/Ser linker, e.g.,(G₄S)_(n), wherein n is 1-10, 2-5, 1, 2, 3, 4, or 5.

In some aspects, the disclosure provides heterodimers which bind type 1interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with or without a linker domain (e.g., a polypeptide linker,e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2,3, 4, or 5) to a mutant Fc domain comprising mutations T350V, T366L,K392L and T394W, and wherein the second polypeptide comprises an IFNAR2domain operatively coupled with or without a linker domain (e.g., apolypeptide linker, e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein nis 1-10, 2-5, 1, 2, 3, 4, or 5) to a mutant Fc domain comprisingmutations T350V, L351Y, F405A and Y407V, according to EU numbering. Insome aspects, the first and second polypeptide each comprise a mutanthuman IgG1 Fc domain. In some aspects, the first and second polypeptideeach comprise a polypeptide linker, wherein the polypeptide linker is aGly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2, 3, 4, or5.

In some aspects, the disclosure provides heterodimers which bind type 1interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with or without a linker domain (e.g., a polypeptide linker,e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2,3, 4, or 5) to a mutant Fc domain comprising mutations T350V, L351Y,F405A and Y407V, and wherein the second polypeptide comprises an IFNAR2domain operatively coupled with or without a linker domain (e.g., apolypeptide linker, e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein nis 1-10, 2-5, 1, 2, 3, 4, or 5) to a mutant Fc domain comprisingmutations T350V, T366L, K392L and T394W, according to EU numbering. Insome aspects, the first and second polypeptide each comprise a mutanthuman IgG1 Fc domain. In some aspects, the first and second polypeptideeach comprise a polypeptide linker, wherein the polypeptide linker is aGly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2, 3, 4, or5.

In some aspects, the disclosure provides heterodimers which bind type 1interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with or without a linker domain (e.g., a polypeptide linker,e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2,3, 4, or 5) to a mutant Fc domain comprising a mutation T366Y, andwherein the second polypeptide comprises an IFNAR2 domain operativelycoupled with or without a linker domain (e.g., a polypeptide linker,e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2,3, 4, or 5) to a mutant Fc domain comprising mutations T366S, L368A andY407V, according to EU numbering. In some aspects, the first and secondpolypeptide each comprise a mutant human IgG4 Fc domain. In someaspects, the first and second polypeptide each comprise a polypeptidelinker, wherein the polypeptide linker is a Gly/Ser linker, e.g.,(G₄S)_(n), wherein n is 1-10, 2-5, 1, 2, 3, 4, or 5.

In some aspects, the disclosure provides heterodimers which bind type 1interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with or without a linker domain (e.g., a polypeptide linker,e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2,3, 4, or 5) to a mutant Fc domain comprising mutations T366S, L368A andY407V, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled with or without a linker domain (e.g., a polypeptidelinker, e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5,1, 2, 3, 4, or 5) to a mutant Fc domain comprising a mutation T366Y,according to EU numbering. In some aspects, the first and secondpolypeptide each comprise a mutant human IgG4 Fc domain. In someaspects, the first and second polypeptide each comprise a polypeptidelinker, wherein the polypeptide linker is a Gly/Ser linker, e.g.,(G₄S)_(n), wherein n is 1-10, 2-5, 1, 2, 3, 4, or 5.

In some aspects, the disclosure provides heterodimers which bind type 1interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with or without a linker domain (e.g., a polypeptide linker,e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2,3, 4, or 5) to a mutant Fc domain comprising mutations T350V, T366L,K392L and T394W, and wherein the second polypeptide comprises an IFNAR2domain operatively coupled with or without a linker domain (e.g., apolypeptide linker, e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein nis 1-10, 2-5, 1, 2, 3, 4, or 5) to a mutant Fc domain comprisingmutations T350V, L351Y, F405A and Y407V, according to EU numbering. Insome aspects, the first and second polypeptide each comprise a mutanthuman IgG4 Fc domain. In some aspects, the first and second polypeptideeach comprise a polypeptide linker, wherein the polypeptide linker is aGly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2, 3, 4, or5.

In some aspects, the disclosure provides heterodimers which bind type 1interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with or without a linker domain (e.g., a polypeptide linker,e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2,3, 4, or 5) to a mutant Fc domain comprising mutations T350V, L351Y,F405A and Y407V, and wherein the second polypeptide comprises an IFNAR2domain operatively coupled with or without a linker domain (e.g., apolypeptide linker, e.g., a Gly/Ser linker, e.g., (G₄S)_(n), wherein nis 1-10, 2-5, 1, 2, 3, 4, or 5) to a mutant Fc domain comprisingmutations T350V, T366L, K392L and T394W, according to EU numbering. Insome aspects, the first and second polypeptide each comprise a mutanthuman IgG4 Fc domain. In some aspects, the first and second polypeptideeach comprise a polypeptide linker, wherein the polypeptide linker is aGly/Ser linker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2, 3, 4, or5.

In any of the foregoing or related aspects, the heterodimer of thedisclosure comprises a first polypeptide comprising an IFNAR1 domain anda second polypeptide comprising an IFNAF2 domain, wherein each of thefirst and second polypeptides is operably coupled with or without alinker to a mutant Fc domain comprising one or more mutations (e.g., oneor more CH2 mutations, one or more CH3 mutations, or one or more CH2 andCH3 mutations), wherein the one or more Fc mutations promotes,increases, or enhances the formation of a heterodimer relative to aheterodimer comprising a first polypeptide comprising an IFNAR1 domainand a second polypeptide comprising an IFNAF2 domain, each comprising awild-type Fc domain (e.g., an Fc domain of the same isotype (e.g., humanIgG1 or human IgG4) without the one or more Fc mutations). In someaspects, the mutant Fc domain is a mutant human IgG1 Fc domain. In someaspects, the mutant Fc domain is a mutant human IgG4 domain

In any of the foregoing or related aspects, the heterodimer of thedisclosure comprises a first polypeptide comprising an IFNAR1 domain anda second polypeptide comprising an IFNAF2 domain, wherein each of thefirst and second polypeptides is operably coupled with or without alinker to a mutant Fc domain comprising one or more mutations (e.g., oneor more CH2 mutations, one or more CH3 mutations, or one or more CH2 andCH3 mutations), wherein the mutant Fc domain of the first polypeptideand/or the mutant Fc domain of the second polypeptide further comprisesone or more mutations which promotes, increases, or enhances stabilityof the Fc domain, and/or reduces binding to Fc receptors. In someaspects, the mutation is selected from the group consisting of: C220S,C226S, C229S, P238S, and P331S, and a combination thereof, according toEU numbering. In some aspects, the mutations comprise C220S, P238S, andP331S. In some aspects, the mutations comprise C220S, C226S, C229S,P238S, and P331S. In some aspects, the first polypeptide and secondpolypeptide each comprise a mutant Fc domain comprising mutations C220S,P238S, and P331S. In some aspects, the first polypeptide and secondpolypeptide each comprise a mutant Fc domain comprising mutations C220S,C226S, C229S, P238S, and P331S.

In any of the foregoing or related aspects, the heterodimer of thedisclosure comprises a first polypeptide comprising an IFNAR1 domain anda second polypeptide comprising an IFNAF2 domain, wherein each of thefirst and second polypeptides is operably coupled with or without alinker to a mutant Fc domain, wherein the first polypeptide comprises anIFNAR1 domain comprising the amino acid sequence in SEQ ID NO: 11 andthe second polypeptide comprises an IFNAR2 domain comprising the aminoacid sequence in SEQ ID NO: 12.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled to a mutant IgG1 Fc domain comprising mutation T366Y, andwherein the second polypeptide comprises an IFNAR2 domain operativelycoupled to a mutant IgG1 Fc domain comprising mutation Y407T, accordingto EU numbering. In some aspects, the first polypeptide comprises anIFNAR1 domain comprising the amino acid sequence in SEQ ID NO: 11 andthe second polypeptide comprises an IFNAR2 domain comprising the aminoacid sequence in SEQ ID NO: 12. In some aspects, the mutant IgG1 Fcdomain comprises the amino acid sequence set forth in SEQ ID NO: 106. Insome aspects, the mutant IgG1 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 107.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled to a mutant IgG1 Fc domain comprising mutation Y407T, andwherein the second polypeptide comprises an IFNAR2 domain operativelycoupled to a mutant IgG1 Fc domain comprising mutation T336Y, accordingto EU numbering. In some aspects, the first polypeptide comprises anIFNAR1 domain comprising the amino acid sequence in SEQ ID NO: 11 andthe second polypeptide comprises an IFNAR2 domain comprising the aminoacid sequence in SEQ ID NO: 12. In some aspects, the mutant IgG1 Fcdomain comprises the amino acid sequence set forth in SEQ ID NO: 106. Insome aspects, the mutant IgG1 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 107.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled to a mutant IgG1 Fc domain comprising mutation T366W, andwherein the second polypeptide comprises an IFNAR2 domain operativelycoupled to a mutant IgG1 Fc domain comprising mutations T366S, L368A andY407V, according to EU numbering. In some aspects, the first polypeptidecomprises an IFNAR1 domain comprising the amino acid sequence in SEQ IDNO: 11 and the second polypeptide comprises an IFNAR2 domain comprisingthe amino acid sequence in SEQ ID NO: 12. In some aspects, the mutantIgG1 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:108. In some aspects, the mutant IgG1 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 109.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled to a mutant IgG1 Fc domain comprising mutations T366S, L368A andY407V, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled to a mutant IgG1 Fc domain comprising mutationT336W, according to EU numbering. In some aspects, the first polypeptidecomprises an IFNAR1 domain comprising the amino acid sequence in SEQ IDNO: 11 and the second polypeptide comprises an IFNAR2 domain comprisingthe amino acid sequence in SEQ ID NO: 12. In some aspects, the mutantIgG1 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:108. In some aspects, the mutant IgG1 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 109.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled to a mutant IgG1 Fc domain comprising mutations T350V, T366L,K392L and T394W, and wherein the second polypeptide comprises an IFNAR2domain operatively coupled to a mutant IgG1 Fc domain comprisingmutations T350V, L351Y, F405A and Y407V, according to EU numbering. Insome aspects, the first polypeptide comprises an IFNAR1 domaincomprising the amino acid sequence in SEQ ID NO: 11 and the secondpolypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG1 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 9. In someaspects, the mutant IgG1 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 10.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled to a mutant IgG1 Fc domain comprising mutations T350V, L351Y,F405A and Y407V, and wherein the second polypeptide comprises an IFNAR2domain operatively coupled to a mutant IgG1 Fc domain comprisingmutations T350V, T366L, K392L and T394W, according to EU numbering. Insome aspects, the first polypeptide comprises an IFNAR1 domaincomprising the amino acid sequence in SEQ ID NO: 11 and the secondpolypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG1 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 9. In someaspects, the mutant IgG1 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 10.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled to a mutant IgG4 Fc domain comprising mutation T366Y, andwherein the second polypeptide comprises an IFNAR2 domain operativelycoupled to a mutant IgG4 Fc domain comprising mutation Y407T, accordingto EU numbering. In some aspects, the first polypeptide comprises anIFNAR1 domain comprising the amino acid sequence in SEQ ID NO: 11 andthe second polypeptide comprises an IFNAR2 domain comprising the aminoacid sequence in SEQ ID NO: 12. In some aspects, the mutant IgG4 Fcdomain comprises the amino acid sequence set forth in SEQ ID NO: 122. Insome aspects, the mutant IgG4 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 123.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled to a mutant IgG4 Fc domain comprising mutation Y407T, andwherein the second polypeptide comprises an IFNAR2 domain operativelycoupled to a mutant IgG4 Fc domain comprising mutation T336Y, accordingto EU numbering. In some aspects, the first polypeptide comprises anIFNAR1 domain comprising the amino acid sequence in SEQ ID NO: 11 andthe second polypeptide comprises an IFNAR2 domain comprising the aminoacid sequence in SEQ ID NO: 12. In some aspects, the mutant IgG4 Fcdomain comprises the amino acid sequence set forth in SEQ ID NO: 122. Insome aspects, the mutant IgG4 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 123.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled to a mutant IgG4 Fc domain comprising mutation T366W, andwherein the second polypeptide comprises an IFNAR2 domain operativelycoupled to a mutant IgG4 Fc domain comprising mutations T366S, L368A andY407V, according to EU numbering. In some aspects, the first polypeptidecomprises an IFNAR1 domain comprising the amino acid sequence in SEQ IDNO: 11 and the second polypeptide comprises an IFNAR2 domain comprisingthe amino acid sequence in SEQ ID NO: 12. In some aspects, the mutantIgG4 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:124. In some aspects, the mutant IgG4 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 125.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled to a mutant IgG4 Fc domain comprising mutations T366S, L368A andY407V, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled to a mutant IgG4 Fc domain comprising mutationT336W, according to EU numbering. In some aspects, the first polypeptidecomprises an IFNAR1 domain comprising the amino acid sequence in SEQ IDNO: 11 and the second polypeptide comprises an IFNAR2 domain comprisingthe amino acid sequence in SEQ ID NO: 12. In some aspects, the mutantIgG4 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:124. In some aspects, the mutant IgG4 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 125.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled to a mutant IgG4 Fc domain comprising mutations T350V, T366L,K392L and T394W, and wherein the second polypeptide comprises an IFNAR2domain operatively coupled to a mutant IgG4 Fc domain comprisingmutations T350V, L351Y, F405A and Y407V, according to EU numbering. Insome aspects, the first polypeptide comprises an IFNAR1 domaincomprising the amino acid sequence in SEQ ID NO: 11 and the secondpolypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG4 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 120. In someaspects, the mutant IgG4 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 121.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled to a mutant IgG4 Fc domain comprising mutations T350V, L351Y,F405A and Y407V, and wherein the second polypeptide comprises an IFNAR2domain operatively coupled to a mutant IgG4 Fc domain comprisingmutations T350V, T366L, K392L and T394W, according to EU numbering. Insome aspects, the first polypeptide comprises an IFNAR1 domaincomprising the amino acid sequence in SEQ ID NO: 11 and the secondpolypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG4 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 120. In someaspects, the mutant IgG4 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 121.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with a Gly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is1-5, 1, 2, 3, 4 or 5) to a mutant IgG1 Fc domain comprising mutationT366Y, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled to a mutant IgG1 Fc domain comprising mutationY407T, according to EU numbering. In some aspects, the first polypeptidecomprises an IFNAR1 domain comprising the amino acid sequence in SEQ IDNO: 11 and the second polypeptide comprises an IFNAR2 domain comprisingthe amino acid sequence in SEQ ID NO: 12. In some aspects, the mutantIgG1 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:106. In some aspects, the mutant IgG1 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 107.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with a Gly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is1-5, 1, 2, 3, 4 or 5) to a mutant IgG1 Fc domain comprising mutationY407T, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled to a mutant IgG1 Fc domain comprising mutationT336Y, according to EU numbering. In some aspects, the first polypeptidecomprises an IFNAR1 domain comprising the amino acid sequence in SEQ IDNO: 11 and the second polypeptide comprises an IFNAR2 domain comprisingthe amino acid sequence in SEQ ID NO: 12. In some aspects, the mutantIgG1 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:106. In some aspects, the mutant IgG1 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 107.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with a Gly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is1-5, 1, 2, 3, 4 or 5) to a mutant IgG1 Fc domain comprising mutationT366W, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled to a mutant IgG1 Fc domain comprising mutationsT366S, L368A and Y407V, according to EU numbering. In some aspects, thefirst polypeptide comprises an IFNAR1 domain comprising the amino acidsequence in SEQ ID NO: 11 and the second polypeptide comprises an IFNAR2domain comprising the amino acid sequence in SEQ ID NO: 12. In someaspects, the mutant IgG1 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 108. In some aspects, the mutant IgG1 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 109.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with a Gly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is1-5, 1, 2, 3, 4 or 5) to a mutant IgG1 Fc domain comprising mutationsT366S, L368A and Y407V, and wherein the second polypeptide comprises anIFNAR2 domain operatively coupled to a mutant IgG1 Fc domain comprisingmutation T336W, according to EU numbering. In some aspects, the firstpolypeptide comprises an IFNAR1 domain comprising the amino acidsequence in SEQ ID NO: 11 and the second polypeptide comprises an IFNAR2domain comprising the amino acid sequence in SEQ ID NO: 12. In someaspects, the mutant IgG1 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 108. In some aspects, the mutant IgG1 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 109.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with a Gly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is1-5, 1, 2, 3, 4 or 5) to a mutant IgG1 Fc domain comprising mutationsT350V, T366L, K392L and T394W, and wherein the second polypeptidecomprises an IFNAR2 domain operatively coupled to a mutant IgG1 Fcdomain comprising mutations T350V, L351Y, F405A and Y407V, according toEU numbering. In some aspects, the first polypeptide comprises an IFNAR1domain comprising the amino acid sequence in SEQ ID NO: 11 and thesecond polypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG1 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 9. In someaspects, the mutant IgG1 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 10.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with a Gly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is1-5, 1, 2, 3, 4 or 5) to a mutant IgG1 Fc domain comprising mutationsT350V, L351Y, F405A and Y407V, and wherein the second polypeptidecomprises an IFNAR2 domain operatively coupled to a mutant IgG1 Fcdomain comprising mutations T350V, T366L, K392L and T394W, according toEU numbering. In some aspects, the first polypeptide comprises an IFNAR1domain comprising the amino acid sequence in SEQ ID NO: 11 and thesecond polypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG1 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 9. In someaspects, the mutant IgG1 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 10.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with a Gly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is1-5, 1, 2, 3, 4 or 5) to a mutant IgG4 Fc domain comprising mutationT366Y, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled to a mutant IgG4 Fc domain comprising mutationY407T, according to EU numbering. In some aspects, the first polypeptidecomprises an IFNAR1 domain comprising the amino acid sequence in SEQ IDNO: 11 and the second polypeptide comprises an IFNAR2 domain comprisingthe amino acid sequence in SEQ ID NO: 12. In some aspects, the mutantIgG4 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:122. In some aspects, the mutant IgG4 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 123.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with a Gly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is1-5, 1, 2, 3, 4 or 5) to a mutant IgG4 Fc domain comprising mutationY407T, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled to a mutant IgG4 Fc domain comprising mutationT336Y, according to EU numbering. In some aspects, the first polypeptidecomprises an IFNAR1 domain comprising the amino acid sequence in SEQ IDNO: 11 and the second polypeptide comprises an IFNAR2 domain comprisingthe amino acid sequence in SEQ ID NO: 12. In some aspects, the mutantIgG4 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:122. In some aspects, the mutant IgG4 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 123.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with a Gly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is1-5, 1, 2, 3, 4 or 5) to a mutant IgG4 Fc domain comprising mutationT366W, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled to a mutant IgG4 Fc domain comprising mutationsT366S, L368A and Y407V, according to EU numbering. In some aspects, thefirst polypeptide comprises an IFNAR1 domain comprising the amino acidsequence in SEQ ID NO: 11 and the second polypeptide comprises an IFNAR2domain comprising the amino acid sequence in SEQ ID NO: 12. In someaspects, the mutant IgG4 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 124. In some aspects, the mutant IgG4 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 125.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with a Gly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is1-5, 1, 2, 3, 4 or 5) to a mutant IgG4 Fc domain comprising mutationsT366S, L368A and Y407V, and wherein the second polypeptide comprises anIFNAR2 domain operatively coupled to a mutant IgG4 Fc domain comprisingmutation T336W, according to EU numbering. In some aspects, the firstpolypeptide comprises an IFNAR1 domain comprising the amino acidsequence in SEQ ID NO: 11 and the second polypeptide comprises an IFNAR2domain comprising the amino acid sequence in SEQ ID NO: 12. In someaspects, the mutant IgG4 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 124. In some aspects, the mutant IgG4 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 125.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with a Gly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is1-5, 1, 2, 3, 4 or 5) to a mutant IgG4 Fc domain comprising mutationsT350V, T366L, K392L and T394W, and wherein the second polypeptidecomprises an IFNAR2 domain operatively coupled to a mutant IgG4 Fcdomain comprising mutations T350V, L351Y, F405A and Y407V, according toEU numbering. In some aspects, the first polypeptide comprises an IFNAR1domain comprising the amino acid sequence in SEQ ID NO: 11 and thesecond polypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG4 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 120. In someaspects, the mutant IgG4 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 121.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with a Gly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is1-5, 1, 2, 3, 4 or 5) to a mutant IgG4 Fc domain comprising mutationsT350V, L351Y, F405A and Y407V, and wherein the second polypeptidecomprises an IFNAR2 domain operatively coupled to a mutant IgG4 Fcdomain comprising mutations T350V, T366L, K392L and T394W, according toEU numbering. In some aspects, the first polypeptide comprises an IFNAR1domain comprising the amino acid sequence in SEQ ID NO: 11 and thesecond polypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG4 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 120. In someaspects, the mutant IgG4 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 121.

In other aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide and a second polypeptideselected from:

(i) a first polypeptide comprising the amino acid sequence of SEQ ID NO:40 and a second polypeptide comprising the amino acid sequence of SEQ IDNO: 43;

(ii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 41 and a second polypeptide comprising the amino acid sequence ofSEQ ID NO: 42;

(iii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 53 and a second polypeptide comprising the amino acid sequence ofSEQ ID NO: 55; and

(iv) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 57 and a second polypeptide comprising the amino acid sequence ofSEQ ID NO: 59;

(v) a first polypeptide comprising the amino acid sequence of SEQ ID NO:40, and a second polypeptide comprising the amino acid sequence selectedfrom SEQ ID NO: 43, SEQ ID NO: 55, and SEQ ID NO: 59;

(vi) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 43, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 40, SEQ ID NO: 53, and SEQ ID NO: 57;

(vii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 42, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 41, SEQ ID NO: 45, and SEQ ID NO: 47;

(viii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 41, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 42, SEQ ID NO: 49, and SEQ ID NO: 51;

(ix) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 53, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 43, SEQ ID NO: 55, and SEQ ID NO: 59;

(x) a first polypeptide comprising the amino acid sequence of SEQ ID NO:55, and a second polypeptide comprising the amino acid sequence selectedfrom SEQ ID NO: 40, SEQ ID NO: 53, and SEQ ID NO: 57;

(xi) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 57, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 43, SEQ ID NO: 55, and SEQ ID NO: 59;

(xii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 59, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 40, SEQ ID NO: 53, and SEQ ID NO: 57;

(xiii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 45, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 42, SEQ ID NO: 49, and SEQ ID NO: 51;

(xiv) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 47, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 42, SEQ ID NO: 49, and SEQ ID NO: 51;

(xv) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 49, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 41, SEQ ID NO: 45, and SEQ ID NO: 47;

(xvi) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 51, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 41, SEQ ID NO: 45, and SEQ ID NO: 47;

(xvii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 61, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 67, SEQ ID NO: 75, and SEQ ID NO: 82;

(xviii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 63, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 65, SEQ ID NO: 73, and SEQ ID NO: 81;

(xix) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 65, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 63, SEQ ID NO: 71, and SEQ ID NO: 79;

(xx) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 67, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 61, SEQ ID NO: 69, and SEQ ID NO: 77;

(xxi) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 69, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 67, SEQ ID NO: 75, and SEQ ID NO: 82;

(xxii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 71, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 65, SEQ ID NO: 73, and SEQ ID NO: 81;

(xxiii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 73, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 63, SEQ ID NO: 71, and SEQ ID NO: 79;

(xxiv) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 75, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 61, SEQ ID NO: 69, and SEQ ID NO: 77;

(xxv) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 77, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 67, SEQ ID NO: 75, and SEQ ID NO: 82;

(xxvi) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 79, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 65, SEQ ID NO: 73, and SEQ ID NO: 81;

(xxvii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 81, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 63, SEQ ID NO: 71, and SEQ ID NO: 79;

(xxviii) a first polypeptide comprising the amino acid sequence of SEQID NO: 82, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 61, SEQ ID NO: 69, and SEQ ID NO: 77;

(xxix) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 84, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 89, SEQ ID NO: 97, and SEQ ID NO: 105;

(xxx) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 85, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 87, SEQ ID NO: 95, and SEQ ID NO: 103;

(xxxi) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 87, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 85, SEQ ID NO: 93, and SEQ ID NO: 101;

(xxxii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 89, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 84, SEQ ID NO: 91, and SEQ ID NO: 99;

(xxxiii) a first polypeptide comprising the amino acid sequence of SEQID NO: 91, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 89, SEQ ID NO: 97, and SEQ ID NO: 105;

(xxxiv) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 93, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 87, SEQ ID NO: 95, and SEQ ID NO: 103;

(xxxv) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 95, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 85, SEQ ID NO: 93, and SEQ ID NO: 101;

(xxxvi) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 97, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 84, SEQ ID NO: 91, and SEQ ID NO: 99;

(xxxvii) a first polypeptide comprising the amino acid sequence of SEQID NO: 99, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 89, SEQ ID NO: 97, and SEQ ID NO: 105;

(xxxviii) a first polypeptide comprising the amino acid sequence of SEQID NO: 101, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 87, SEQ ID NO: 95, and SEQ ID NO: 103;

(xxxix) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 103, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 85, SEQ ID NO: 93, and SEQ ID NO: 101; and

(xl) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 105, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 84, SEQ ID NO: 91, and SEQ ID NO: 99.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide comprising the amino acidsequence of SEQ ID NO: 40 and a second polypeptide comprising the aminoacid sequence of SEQ ID NO: 43.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide comprising the amino acidsequence of SEQ ID NO: 41 and a second polypeptide comprising the aminoacid sequence of SEQ ID NO: 42.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide comprising the amino acidsequence of SEQ ID NO: 53 and a second polypeptide comprising the aminoacid sequence of SEQ ID NO: 55.

In some aspects, the disclosure provides a heterodimer which binds typeI interferons comprising a first polypeptide comprising the amino acidsequence of SEQ ID NO: 57 and a second polypeptide comprising the aminoacid sequence of SEQ ID NO: 59.

In any of the foregoing or related aspects, the disclosure provides aheterodimer which binds type 1 interferons, wherein the type Iinterferons are selected from interferon-α (INFα), interferon-β (INFβ),or both INFα and INFβ. In some aspects, the type I interferon is INFα.In some aspects, the type I interferon is INFβ. In some aspects, thetype I interferon is both INFα and INFβ. In some aspects, theheterodimer inhibits an activity of INFα. In some aspects, theheterodimer inhibits an activity of INFβ. In some aspects, theheterodimer inhibits an activity of both INFα and INFβ. In some aspects,the heterodimer inhibits induction of type I interferon (IFN) geneexpression.

In any of the foregoing or related aspects, the disclosure provides aheterodimer which binds type 1 interferons comprising a first and secondpolypeptide, wherein each of the first and second polypeptides is linkeddirectly to a mutant Fc domain (without a linker), and wherein theheterodimer has increased binding to type I interferons relative to aheterodimer in which each of the first and second polypeptides comprisea polypeptide linker domain.

In any of the foregoing or related aspects, the disclosure provides aheterodimer which binds type 1 interferons comprising a firstpolypeptide comprising an IFNAR1 domain and a second polypeptidecomprising an IFNAF2 domain, wherein each of the first and secondpolypeptides is operably coupled to a mutant Fc domain comprising one ormore mutations (e.g., one or more CH2 mutations, one or more CH3mutations, or one or more CH2 and CH3 mutations), and wherein the one ormore Fc mutations promotes, increases, or enhances the formation of aheterodimer relative to a heterodimer comprising a first polypeptidecomprising an IFNAR1 domain and a second polypeptide comprising anIFNAF2 domain, each comprising a wild-type Fc domain.

In any of the foregoing or related aspects, the heterodimer of thedisclosure comprises a first polypeptide comprising an IFNAR1 domain anda second polypeptide comprising an IFNAF2 domain, wherein each of thefirst and second polypeptides is operably coupled with or without alinker to a mutant Fc domain comprising one or more mutations (e.g., oneor more CH2 mutations, one or more CH3 mutations, or one or more CH2 andCH3 mutations), wherein the mutant Fc domain of the first polypeptideand/or the mutant Fc domain of the second polypeptide further comprisesone or more mutations which promotes, increases, or enhances stabilityof the Fc domain, and/or reduces binding to Fc receptors. In someaspects, the mutation is selected from the group consisting of: C220S,C226S, C229S, P238S, and P331S, and a combination thereof, according toEU numbering. In some aspects, the mutations comprise C220S, P238S, andP331S. In some aspects, the mutations comprise C220S, C226S, C229S,P238S, and P331S. In some aspects, the first polypeptide and secondpolypeptide each comprise a mutant Fc domain comprising mutations C220S,P238S, and P331S. In some aspects, the first polypeptide and secondpolypeptide each comprise a mutant Fc domain comprising mutations C220S,C226S, C229S, P238S, and P331S.

In other aspects, the disclosure provides a composition comprising aheterodimer of the disclosure and a pharmaceutically acceptable carrier.

In other aspects, the disclosure provides a nucleic acid comprising anucleotide sequence encoding a first polypeptide of the heterodimer,wherein the first polypeptide comprises an IFNAR1 domain operativelycoupled with or without a linker (e.g., a Gly/Ser linker (e.g., a(G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4 or 5) to a mutant Fcdomain (e.g., a mutant human IgG1 Fc domain or a mutant human IgG4 Fcdomain). In some aspects, the first polypeptide comprising an IFNAR1domain comprises the amino acid sequence in SEQ ID NO: 11.

In other aspects, the disclosure provides a nucleic acid comprising anucleotide sequence encoding a second polypeptide of the heterodimer,wherein the second polypeptide comprises an IFNAR2 domain operativelycoupled with or without a linker (e.g., a Gly/Ser linker (e.g., a(G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4 or 5) to a mutant Fcdomain (e.g., a mutant human IgG1 Fc domain or a mutant human IgG4 Fcdomain). In some aspects, the second polypeptide comprising an IFNAR2domain comprises the amino acid sequence in SEQ ID NO: 12.

Other aspects of the disclosure provide recombinant expression vectorsand host cells comprising the nucleic acids of the disclosure. In someaspects, the recombinant expression vector and host cell comprises anucleic acid comprising a nucleotide sequence encoding a firstpolypeptide of the heterodimer, wherein the first polypeptide comprisesan IFNAR1 domain operatively coupled with or without a linker (e.g., aGly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4or 5) to a mutant Fc domain (e.g., a mutant human IgG1 Fc domain or amutant human IgG4 Fc domain). In some aspects, the recombinantexpression vector and host cell comprises a nucleic acid comprising anucleotide sequence encoding a second polypeptide of the heterodimer,wherein the second polypeptide comprises an IFNAR2 domain operativelycoupled with or without a linker (e.g., a Gly/Ser linker (e.g., a(G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4 or 5) to a mutant Fcdomain (e.g., a mutant human IgG1 Fc domain or a mutant human IgG4 Fcdomain). In some aspects, the recombinant expression vector and hostcell comprises both a nucleic acid comprising a nucleotide sequenceencoding the first polypeptide of the heterodimer and a nucleic acidcomprising a nucleotide sequence encoding the a second polypeptide ofthe heterodimer. In some aspects, the first polypeptide comprising anIFNAR1 domain comprises the amino acid sequence in SEQ ID NO: 11. Insome aspects, the second polypeptide comprising an IFNAR2 domaincomprises the amino acid sequence in SEQ ID NO: 12.

Other aspects of the disclosure provide methods of reducing, decreasing,or inhibiting interferon (IFN) gene expression in a subject in needthereof, comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier.

Other aspects of the disclosure provide methods of reducing, decreasing,or inhibiting type I interferons in a subject in need thereof,comprising administering to the subject a heterodimer of the disclosure,or a composition of the disclosure comprising a heterodimer and apharmaceutically acceptable carrier. In some aspects, the type Iinterferon is INFα. In some aspects, the type I interferon is INFβ. Insome aspects, the type I interferon is both INFα and INFβ.

Other aspects of the disclosure provide methods of treating orinhibiting progression of a disease characterized by type I interferonsand methods of treating an autoimmune disease in a subject in needthereof, comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier. In some aspects, the type Iinterferon is INFα. In some aspects, the type I interferon is INFβ. Insome aspects, the type I interferon is both INFα and INFβ. In someaspects, the autoimmune disease is SLE. In some aspects, the autoimmunedisease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ), and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled to amutant IgG1 Fc domain comprising mutation T366Y, and wherein the secondpolypeptide comprises an IFNAR2 domain operatively coupled to a mutantIgG1 Fc domain comprising mutation Y407T, according to EU numbering. Insome aspects, the first polypeptide comprises an IFNAR1 domaincomprising the amino acid sequence in SEQ ID NO: 11 and the secondpolypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG1 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 106. In someaspects, the mutant IgG1 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 107. In some aspects, the autoimmune disease is SLE.In some aspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ), and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled to amutant IgG1 Fc domain comprising mutation Y407T, and wherein the secondpolypeptide comprises an IFNAR2 domain operatively coupled to a mutantIgG1 Fc domain comprising mutation T336Y, according to EU numbering. Insome aspects, the first polypeptide comprises an IFNAR1 domaincomprising the amino acid sequence in SEQ ID NO: 11 and the secondpolypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG1 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 106. In someaspects, the mutant IgG1 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 107. In some aspects, the autoimmune disease is SLE.In some aspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ), and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled to amutant IgG1 Fc domain comprising mutation T366W, and wherein the secondpolypeptide comprises an IFNAR2 domain operatively coupled to a mutantIgG1 Fc domain comprising mutations T366S, L368A and Y407V, according toEU numbering. In some aspects, the first polypeptide comprises an IFNAR1domain comprising the amino acid sequence in SEQ ID NO: 11 and thesecond polypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG1 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 108. In someaspects, the mutant IgG1 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 109. In some aspects, the autoimmune disease is SLE.In some aspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ), and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled to amutant IgG1 Fc domain comprising mutations T366S, L368A and Y407V, andwherein the second polypeptide comprises an IFNAR2 domain operativelycoupled to a mutant IgG1 Fc domain comprising mutation T336W, accordingto EU numbering. In some aspects, the first polypeptide comprises anIFNAR1 domain comprising the amino acid sequence in SEQ ID NO: 11 andthe second polypeptide comprises an IFNAR2 domain comprising the aminoacid sequence in SEQ ID NO: 12. In some aspects, the mutant IgG1 Fcdomain comprises the amino acid sequence set forth in SEQ ID NO: 108. Insome aspects, the mutant IgG1 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 109. In some aspects, the autoimmunedisease is SLE. In some aspects, the autoimmune disease is Sjogren'ssyndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ), and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled to amutant IgG1 Fc domain comprising mutations T350V, T366L, K392L andT394W, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled to a mutant IgG1 Fc domain comprising mutationsT350V, L351Y, F405A and Y407V, according to EU numbering. In someaspects, the first polypeptide comprises an IFNAR1 domain comprising theamino acid sequence in SEQ ID NO: 11 and the second polypeptidecomprises an IFNAR2 domain comprising the amino acid sequence in SEQ IDNO: 12. In some aspects, the mutant IgG1 Fc domain comprises the aminoacid sequence set forth in SEQ ID NO: 9. In some aspects, the mutantIgG1 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:10. In some aspects, the autoimmune disease is SLE. In some aspects, theautoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ), and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled to amutant IgG1 Fc domain comprising mutations T350V, L351Y, F405A andY407V, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled to a mutant IgG1 Fc domain comprising mutationsT350V, T366L, K392L and T394W, according to EU numbering. In someaspects, the first polypeptide comprises an IFNAR1 domain comprising theamino acid sequence in SEQ ID NO: 11 and the second polypeptidecomprises an IFNAR2 domain comprising the amino acid sequence in SEQ IDNO: 12. In some aspects, the mutant IgG1 Fc domain comprises the aminoacid sequence set forth in SEQ ID NO: 9. In some aspects, the mutantIgG1 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:10. In some aspects, the autoimmune disease is SLE. In some aspects, theautoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ), and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled to amutant IgG4 Fc domain comprising mutation T366Y, and wherein the secondpolypeptide comprises an IFNAR2 domain operatively coupled to a mutantIgG4 Fc domain comprising mutation Y407T, according to EU numbering. Insome aspects, the first polypeptide comprises an IFNAR1 domaincomprising the amino acid sequence in SEQ ID NO: 11 and the secondpolypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG4 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 122. In someaspects, the mutant IgG4 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 123. In some aspects, the autoimmune disease is SLE.In some aspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ), and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled to amutant IgG4 Fc domain comprising mutation Y407T, and wherein the secondpolypeptide comprises an IFNAR2 domain operatively coupled to a mutantIgG4 Fc domain comprising mutation T336Y, according to EU numbering. Insome aspects, the first polypeptide comprises an IFNAR1 domaincomprising the amino acid sequence in SEQ ID NO: 11 and the secondpolypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG4 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 122. In someaspects, the mutant IgG4 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 123. In some aspects, the autoimmune disease is SLE.In some aspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ), and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled to amutant IgG4 Fc domain comprising mutation T366W, and wherein the secondpolypeptide comprises an IFNAR2 domain operatively coupled to a mutantIgG4 Fc domain comprising mutations T366S, L368A and Y407V, according toEU numbering. In some aspects, the first polypeptide comprises an IFNAR1domain comprising the amino acid sequence in SEQ ID NO: 11 and thesecond polypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG4 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 124. In someaspects, the mutant IgG4 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 125. In some aspects, the autoimmune disease is SLE.In some aspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ), and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled to amutant IgG4 Fc domain comprising mutations T366S, L368A and Y407V, andwherein the second polypeptide comprises an IFNAR2 domain operativelycoupled to a mutant IgG4 Fc domain comprising mutation T336W, accordingto EU numbering. In some aspects, the first polypeptide comprises anIFNAR1 domain comprising the amino acid sequence in SEQ ID NO: 11 andthe second polypeptide comprises an IFNAR2 domain comprising the aminoacid sequence in SEQ ID NO: 12. In some aspects, the mutant IgG4 Fcdomain comprises the amino acid sequence set forth in SEQ ID NO: 124. Insome aspects, the mutant IgG4 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 125. In some aspects, the autoimmunedisease is SLE. In some aspects, the autoimmune disease is Sjogren'ssyndrome. In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ), and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled to amutant IgG4 Fc domain comprising mutations T350V, T366L, K392L andT394W, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled to a mutant IgG4 Fc domain comprising mutationsT350V, L351Y, F405A and Y407V, according to EU numbering. In someaspects, the first polypeptide comprises an IFNAR1 domain comprising theamino acid sequence in SEQ ID NO: 11 and the second polypeptidecomprises an IFNAR2 domain comprising the amino acid sequence in SEQ IDNO: 12. In some aspects, the mutant IgG4 Fc domain comprises the aminoacid sequence set forth in SEQ ID NO: 120. In some aspects, the mutantIgG4 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:121. In some aspects, the autoimmune disease is SLE. In some aspects,the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ), and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled to amutant IgG4 Fc domain comprising mutations T350V, L351Y, F405A andY407V, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled to a mutant IgG4 Fc domain comprising mutationsT350V, T366L, K392L and T394W, according to EU numbering. In someaspects, the first polypeptide comprises an IFNAR1 domain comprising theamino acid sequence in SEQ ID NO: 11 and the second polypeptidecomprises an IFNAR2 domain comprising the amino acid sequence in SEQ IDNO: 12. In some aspects, the mutant IgG4 Fc domain comprises the aminoacid sequence set forth in SEQ ID NO: 120. In some aspects, the mutantIgG4 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:121. In some aspects, the autoimmune disease is SLE. In some aspects,the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled with aGly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4or 5) to a mutant IgG1 Fc domain comprising mutation T366Y, and whereinthe second polypeptide comprises an IFNAR2 domain operatively coupled toa mutant IgG1 Fc domain comprising mutation Y407T, according to EUnumbering. In some aspects, the first polypeptide comprises an IFNAR1domain comprising the amino acid sequence in SEQ ID NO: 11 and thesecond polypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG1 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 106. In someaspects, the mutant IgG1 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 107. In some aspects, the autoimmune disease is SLE.In some aspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled with aGly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4or 5) to a mutant IgG1 Fc domain comprising mutation Y407T, and whereinthe second polypeptide comprises an IFNAR2 domain operatively coupled toa mutant IgG1 Fc domain comprising mutation T336Y, according to EUnumbering. In some aspects, the first polypeptide comprises an IFNAR1domain comprising the amino acid sequence in SEQ ID NO: 11 and thesecond polypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG1 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 106. In someaspects, the mutant IgG1 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 107. In some aspects, the autoimmune disease is SLE.In some aspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled with aGly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4or 5) to a mutant IgG1 Fc domain comprising mutation T366W, and whereinthe second polypeptide comprises an IFNAR2 domain operatively coupled toa mutant IgG1 Fc domain comprising mutations T366S, L368A and Y407V,according to EU numbering. In some aspects, the first polypeptidecomprises an IFNAR1 domain comprising the amino acid sequence in SEQ IDNO: 11 and the second polypeptide comprises an IFNAR2 domain comprisingthe amino acid sequence in SEQ ID NO: 12. In some aspects, the mutantIgG1 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:108. In some aspects, the mutant IgG1 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 109. In some aspects, the autoimmunedisease is SLE. In some aspects, the autoimmune disease is Sjogren'ssyndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled with aGly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4or 5) to a mutant IgG1 Fc domain comprising mutations T366S, L368A andY407V, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled to a mutant IgG1 Fc domain comprising mutationT336W, according to EU numbering. In some aspects, the first polypeptidecomprises an IFNAR1 domain comprising the amino acid sequence in SEQ IDNO: 11 and the second polypeptide comprises an IFNAR2 domain comprisingthe amino acid sequence in SEQ ID NO: 12. In some aspects, the mutantIgG1 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:108. In some aspects, the mutant IgG1 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 109. In some aspects, the autoimmunedisease is SLE. In some aspects, the autoimmune disease is Sjogren'ssyndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled with aGly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4or 5) to a mutant IgG1 Fc domain comprising mutations T350V, T366L,K392L and T394W, and wherein the second polypeptide comprises an IFNAR2domain operatively coupled to a mutant IgG1 Fc domain comprisingmutations T350V, L351Y, F405A and Y407V, according to EU numbering. Insome aspects, the first polypeptide comprises an IFNAR1 domaincomprising the amino acid sequence in SEQ ID NO: 11 and the secondpolypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG1 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 9. In someaspects, the mutant IgG1 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 10. In some aspects, the autoimmune disease is SLE.In some aspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled with aGly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4or 5) to a mutant IgG1 Fc domain comprising mutations T350V, L351Y,F405A and Y407V, and wherein the second polypeptide comprises an IFNAR2domain operatively coupled to a mutant IgG1 Fc domain comprisingmutations T350V, T366L, K392L and T394W, according to EU numbering. Insome aspects, the first polypeptide comprises an IFNAR1 domaincomprising the amino acid sequence in SEQ ID NO: 11 and the secondpolypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG1 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 9. In someaspects, the mutant IgG1 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 10. In some aspects, the autoimmune disease is SLE.In some aspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled with aGly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4or 5) to a mutant IgG4 Fc domain comprising mutation T366Y, and whereinthe second polypeptide comprises an IFNAR2 domain operatively coupled toa mutant IgG4 Fc domain comprising mutation Y407T, according to EUnumbering. In some aspects, the first polypeptide comprises an IFNAR1domain comprising the amino acid sequence in SEQ ID NO: 11 and thesecond polypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG4 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 122. In someaspects, the mutant IgG4 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 123. In some aspects, the autoimmune disease is SLE.In some aspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled with aGly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4or 5) to a mutant IgG4 Fc domain comprising mutation Y407T, and whereinthe second polypeptide comprises an IFNAR2 domain operatively coupled toa mutant IgG4 Fc domain comprising mutation T336Y, according to EUnumbering. In some aspects, the first polypeptide comprises an IFNAR1domain comprising the amino acid sequence in SEQ ID NO: 11 and thesecond polypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG4 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 122. In someaspects, the mutant IgG4 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 123. In some aspects, the autoimmune disease is SLE.In some aspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled with aGly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4or 5) to a mutant IgG4 Fc domain comprising mutation T366W, and whereinthe second polypeptide comprises an IFNAR2 domain operatively coupled toa mutant IgG4 Fc domain comprising mutations T366S, L368A and Y407V,according to EU numbering. In some aspects, the first polypeptidecomprises an IFNAR1 domain comprising the amino acid sequence in SEQ IDNO: 11 and the second polypeptide comprises an IFNAR2 domain comprisingthe amino acid sequence in SEQ ID NO: 12. In some aspects, the mutantIgG4 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:124. In some aspects, the mutant IgG4 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 125. In some aspects, the autoimmunedisease is SLE. In some aspects, the autoimmune disease is Sjogren'ssyndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled with aGly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4or 5) to a mutant IgG4 Fc domain comprising mutations T366S, L368A andY407V, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled to a mutant IgG4 Fc domain comprising mutationT336W, according to EU numbering. In some aspects, the first polypeptidecomprises an IFNAR1 domain comprising the amino acid sequence in SEQ IDNO: 11 and the second polypeptide comprises an IFNAR2 domain comprisingthe amino acid sequence in SEQ ID NO: 12. In some aspects, the mutantIgG4 Fc domain comprises the amino acid sequence set forth in SEQ ID NO:124. In some aspects, the mutant IgG4 Fc domain comprises the amino acidsequence set forth in SEQ ID NO: 125. In some aspects, the autoimmunedisease is SLE. In some aspects, the autoimmune disease is Sjogren'ssyndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled with aGly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4or 5) to a mutant IgG4 Fc domain comprising mutations T350V, T366L,K392L and T394W, and wherein the second polypeptide comprises an IFNAR2domain operatively coupled to a mutant IgG4 Fc domain comprisingmutations T350V, L351Y, F405A and Y407V, according to EU numbering. Insome aspects, the first polypeptide comprises an IFNAR1 domaincomprising the amino acid sequence in SEQ ID NO: 11 and the secondpolypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG4 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 120. In someaspects, the mutant IgG4 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 121. In some aspects, the autoimmune disease is SLE.In some aspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises an IFNAR1 domain operatively coupled with aGly/Ser linker (e.g., a (G₄S)_(n) linker, wherein n is 1-5, 1, 2, 3, 4or 5) to a mutant IgG4 Fc domain comprising mutations T350V, L351Y,F405A and Y407V, and wherein the second polypeptide comprises an IFNAR2domain operatively coupled to a mutant IgG4 Fc domain comprisingmutations T350V, T366L, K392L and T394W, according to EU numbering. Insome aspects, the first polypeptide comprises an IFNAR1 domaincomprising the amino acid sequence in SEQ ID NO: 11 and the secondpolypeptide comprises an IFNAR2 domain comprising the amino acidsequence in SEQ ID NO: 12. In some aspects, the mutant IgG4 Fc domaincomprises the amino acid sequence set forth in SEQ ID NO: 120. In someaspects, the mutant IgG4 Fc domain comprises the amino acid sequence setforth in SEQ ID NO: 121. In some aspects, the autoimmune disease is SLE.In some aspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide and a second polypeptide selected from:

(i) a first polypeptide comprising the amino acid sequence of SEQ ID NO:40 and a second polypeptide comprising the amino acid sequence of SEQ IDNO: 43;

(ii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 41 and a second polypeptide comprising the amino acid sequence ofSEQ ID NO: 42;

(iii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 53 and a second polypeptide comprising the amino acid sequence ofSEQ ID NO: 55; and

(iv) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 57 and a second polypeptide comprising the amino acid sequence ofSEQ ID NO: 59;

(v) a first polypeptide comprising the amino acid sequence of SEQ ID NO:40, and a second polypeptide comprising the amino acid sequence selectedfrom SEQ ID NO: 43, SEQ ID NO: 55, and SEQ ID NO: 59;

(vi) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 43, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 40, SEQ ID NO: 53, and SEQ ID NO: 57;

(vii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 42, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 41, SEQ ID NO: 45, and SEQ ID NO: 47;

(viii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 41, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 42, SEQ ID NO: 49, and SEQ ID NO: 51;

(ix) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 53, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 43, SEQ ID NO: 55, and SEQ ID NO: 59;

(x) a first polypeptide comprising the amino acid sequence of SEQ ID NO:55, and a second polypeptide comprising the amino acid sequence selectedfrom SEQ ID NO: 40, SEQ ID NO: 53, and SEQ ID NO: 57;

(xi) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 57, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 43, SEQ ID NO: 55, and SEQ ID NO: 59;

(xii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 59, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 40, SEQ ID NO: 53, and SEQ ID NO: 57;

(xiii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 45, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 42, SEQ ID NO: 49, and SEQ ID NO: 51;

(xiv) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 47, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 42, SEQ ID NO: 49, and SEQ ID NO: 51;

(xv) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 49, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 41, SEQ ID NO: 45, and SEQ ID NO: 47;

(xvi) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 51, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 41, SEQ ID NO: 45, and SEQ ID NO: 47;

(xvii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 61, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 67, SEQ ID NO: 75, and SEQ ID NO: 82;

(xviii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 63, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 65, SEQ ID NO: 73, and SEQ ID NO: 81;

(xix) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 65, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 63, SEQ ID NO: 71, and SEQ ID NO: 79;

(xx) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 67, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 61, SEQ ID NO: 69, and SEQ ID NO: 77;

(xxi) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 69, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 67, SEQ ID NO: 75, and SEQ ID NO: 82;

(xxii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 71, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 65, SEQ ID NO: 73, and SEQ ID NO: 81;

(xxiii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 73, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 63, SEQ ID NO: 71, and SEQ ID NO: 79;

(xxiv) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 75, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 61, SEQ ID NO: 69, and SEQ ID NO: 77;

(xxv) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 77, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 67, SEQ ID NO: 75, and SEQ ID NO: 82;

(xxvi) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 79, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 65, SEQ ID NO: 73, and SEQ ID NO: 81;

(xxvii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 81, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 63, SEQ ID NO: 71, and SEQ ID NO: 79;

(xxviii) a first polypeptide comprising the amino acid sequence of SEQID NO: 82, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 61, SEQ ID NO: 69, and SEQ ID NO: 77;

(xxix) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 84, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 89, SEQ ID NO: 97, and SEQ ID NO: 105;

(xxx) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 85, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 87, SEQ ID NO: 95, and SEQ ID NO: 103;

(xxxi) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 87, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 85, SEQ ID NO: 93, and SEQ ID NO: 101;

(xxxii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 89, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 84, SEQ ID NO: 91, and SEQ ID NO: 99;

(xxxiii) a first polypeptide comprising the amino acid sequence of SEQID NO: 91, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 89, SEQ ID NO: 97, and SEQ ID NO: 105;

(xxxiv) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 93, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 87, SEQ ID NO: 95, and SEQ ID NO: 103;

(xxxv) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 95, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 85, SEQ ID NO: 93, and SEQ ID NO: 101;

(xxxvi) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 97, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 84, SEQ ID NO: 91, and SEQ ID NO: 99;

(xxxvii) a first polypeptide comprising the amino acid sequence of SEQID NO: 99, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 89, SEQ ID NO: 97, and SEQ ID NO: 105;

(xxxviii) a first polypeptide comprising the amino acid sequence of SEQID NO: 101, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 87, SEQ ID NO: 95, and SEQ ID NO: 103;

(xxxix) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 103, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 85, SEQ ID NO: 93, and SEQ ID NO: 101; and

(xl) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 105, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 84, SEQ ID NO: 91, and SEQ ID NO: 99. In someaspects, the autoimmune disease is SLE. In some aspects, the autoimmunedisease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide comprising the amino acid sequence of SEQID NO: 40 and a second polypeptide comprising the amino acid sequence ofSEQ ID NO: 43. In some aspects, the autoimmune disease is SLE. In someaspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide comprising the amino acid sequence of SEQID NO: 41 and a second polypeptide comprising the amino acid sequence ofSEQ ID NO: 42. In some aspects, the autoimmune disease is SLE. In someaspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide comprising the amino acid sequence of SEQID NO: 53 and a second polypeptide comprising the amino acid sequence ofSEQ ID NO: 55. In some aspects, the autoimmune disease is SLE. In someaspects, the autoimmune disease is Sjogren's syndrome.

In some aspects, the disclosure provides methods of reducing,decreasing, or inhibiting interferon (IFN) gene expression, methods ofreducing, decreasing, or inhibiting type I interferons (e.g., INFα,INFβ, or both INFα and INFβ, and methods of treating an autoimmunedisease (e.g., SLE, Sjogren's syndrome) in a subject in need thereof,the method comprising administering to the subject a heterodimer of thedisclosure, or a composition of the disclosure comprising a heterodimerand a pharmaceutically acceptable carrier, wherein the heterodimercomprises a first polypeptide comprising the amino acid sequence of SEQID NO: 57 and a second polypeptide comprising the amino acid sequence ofSEQ ID NO: 59. In some aspects, the autoimmune disease is SLE. In someaspects, the autoimmune disease is Sjogren's syndrome.

Other aspects of the disclosure provide use of a heterodimer of thedisclosure, and an optional pharmaceutically acceptable carrier, in themanufacture of a medicament for treating or delaying progression of anautoimmune disease in a subject in need thereof, wherein the treatmentcomprises administration of the medicament to a subject in need thereof.

Other aspects of the disclosure provide kits comprising a medicamentcomprising a heterodimer of the disclosure, and an optionalpharmaceutically acceptable carrier, and a package insert comprisinginstructions for administration of the medicament for treating ordelaying progression of an autoimmune disease in a subject in needthereof.

In some aspects, the disclosure provides a kit comprising a containercomprising a heterodimer of the disclosure, and an optionalpharmaceutically acceptable carrier, and a package insert comprisinginstructions for administration of the heterodimer for treating ordelaying progression of an autoimmune disease in a subject in needthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawing, where:

FIG. 1 depicts exemplary soluble interferon receptors RSLV 601-604, RSLV602-603, RSLV 606-611, and RSLV 608-613.

FIG. 2 graphically depicts the inhibition of IFNα induced SEAPproduction in HEK-Blue α/β cells by the inhibitors RSLV 601-604, RSLV602-603, and anti-human IFNα positive control. Human IgG was used as anegative control.

FIG. 3 graphically depicts the inhibition of IFNβ induced SEAPproduction in HEK-Blue α/β cells by the inhibitors RSLV 601-604, RSLV602-603, and anti-human IFNβ positive control. Human IgG was used as anegative control.

FIG. 4 graphically depicts the impact of linker length on the inhibitionof IFNα induced SEAP production in HEK-Blue α/β cells.

FIG. 5 graphically depicts the impact of linker length on the inhibitionof IFNβ induced SEAP production in HEK-Blue α/β cells.

FIG. 6 depicts the inhibition of SLE sera induced interferon geneexpression in PBMC with soluble interferon receptor RSLV 601-604.

FIG. 7 depicts the inhibition of SLE sera induced interferon geneexpression in PBMC with soluble interferon receptor RSLV 608-613.

DETAILED DESCRIPTION

The present disclosure provides soluble heterodimers which bind type Iinterferons and methods of reducing, decreasing, or inhibitinginterferon (IFN) gene expression, methods of reducing, decreasing, orinhibiting type I interferons and/or type I interferon activity (e.g.,INFα, INFβ, or both INFα and INFβ, and methods of treating diseasescharacterized by type I interferon production in a subject in needthereof.

The disclosure is based, at least in part, on the discovery thatheterodimers comprising an IFNAR1 domain and an IFNAR2 domain, eachoperatively coupled to a mutant Fc domain, potently inhibit IFNα andIFNβ activity and inhibit SLE sera induced interferon gene expression.These results indicate that the heterodimers of the disclosure arecapable of binding to and inhibiting the activity of type I interferonsin a subject with an autoimmune condition, such as SLE.

It was also discovered that heterodimers comprising an IFNAR1 domain andan IFNAR2 domain, each operatively coupled directly to a mutant Fcdomain (i.e., without a polypeptide linker) have increased bindingaffinity and potency for type I interferons, such as INFα, relative to aheterodimer in which the IFNAR1 domain and IFNAR2 domain are eachoperatively coupled to a mutant Fc domain with a polypeptide linker.Without being bound by theory, it was believed that IFNAR1 and IFNAR2required a degree of flexibility to form a pocket for interacting with atype I interferons. It was known that the extracellular domains ofIFNAR1 and IFNAR2 bind type I interferons and form a ternary complex,which results in bringing the intracellular domains into close proximityand regulates signaling through the receptor (Li et al., J. Mol. Biol.(2017) 429, 2571-2589). It has also been shown that ligand inducedconformation changes to the receptor components are propagated to themembrane-proximal Ig domain of IFNAR1, although this domain does notinteract with the ligand. (Id. at 2572). Notably, the crystal structureof a heterotrimeric complex of IFNAR1, IFNAR2, and IFNα2 depicts alinker-like domain between the extracellular domain and thetransmembrane domain of both IFNARs. (See Li et al., FIG. 1; also seePiehler et al., Immunological Reviews (2012) 250, 317-334, FIG. 5).Without being bound by theory, it was hypothesized that this linker-likedomain may provide a degree of flexibility to facilitate formation ofthe ternary complex in an appropriate conformation for ligand bindingand signaling. Therefore, a study was designed to investigate the effectof various polypeptide linker lengths on IFNAR1 and IFNAR2 ligandbinding. Unexpectedly, it was discovered that a heterodimer comprisingan IFNAR1 domain and an IFNAR2 domain, each coupled directly to a mutantFc domain, without a linker, showed increased binding affinity andpotency to type I interferon, relative to a heterodimer in which theIFNAR1 domain and IFNAR2 domain are each operatively coupled to a mutantFc domain with a polypeptide linker (e.g., of 10 or 20 amino acids inlength). In particular, when constructs with different polypeptidelinker lengths were compared, it was observed that IFN-α bindingaffinity and potency increased as linker length decreased.

Accordingly, the present disclosure provides heterodimers which bindstype I interferons comprising a first polypeptide and a secondpolypeptide, wherein the first polypeptide comprises an IFNAR1 domainoperatively coupled with or without a linker domain to a mutant Fcdomain, and wherein the second polypeptide comprises an IFNAR2 domainoperatively coupled with or without a linker domain to a mutant Fcdomain. The heterodimers of the disclosure are useful in methods ofinhibiting type I interferon gene expression, methods of inhibiting typeI interferon activity, and methods of treating autoimmune disease, asdescribed herein.

Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that function in amanner similar to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and 0-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, i.e., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs have modified R groups (e.g., norleucine) or modified peptidebackbones, but retain the same basic chemical structure as a naturallyoccurring amino acid. Amino acid mimetics refer to chemical compoundsthat have a structure that is different from the general chemicalstructure of an amino acid, but that function in a manner similar to anaturally occurring amino acid.

Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,can be referred to by their commonly accepted single-letter codes.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence (anamino acid sequence of a starting polypeptide) with a second, different“replacement” amino acid residue. An “amino acid insertion” refers tothe incorporation of at least one additional amino acid into apredetermined amino acid sequence. While the insertion will usuallyconsist of the insertion of one or two amino acid residues, larger“peptide insertions” can be made, e.g. insertion of about three to aboutfive or even up to about ten, fifteen, or twenty amino acid residues.The inserted residue(s) may be naturally occurring or non-naturallyoccurring as disclosed above. An “amino acid deletion” refers to theremoval of at least one amino acid residue from a predetermined aminoacid sequence.

“Polypeptide,” “peptide,” and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid and are metabolized in a mannersimilar to naturally occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences, as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions canbe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res 1991; 19:5081;Ohtsuka et al., JBC 1985; 260:2605-8); Rossolini et al., Mol Cell Probes1994; 8:91-8). For arginine and leucine, modifications at the secondbase can also be conservative. The term nucleic acid is usedinterchangeably with gene, cDNA, and mRNA encoded by a gene.

Polynucleotides of the present invention can be composed of anypolyribonucleotide or polydeoxribonucleotide, which can be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that can be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide can also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

As used herein, the term “operably linked” or “operably coupled” refersto a juxtaposition wherein the components described are in arelationship permitting them to function in their intended manner.

As used herein, the term “glycosylation” or “glycosylated” refers to aprocess or result of adding sugar moieties to a molecule.

As used herein, the term “altered glycosylation” refers to a moleculethat is aglycosylated, deglycosylated, or underglycosylated.

As used herein, “glycosylation site(s)” refers to both sites thatpotentially could accept a carbohydrate moiety, as well as sites withinthe protein on which a carbohydrate moiety has actually been attachedand includes any amino acid sequence that could act as an acceptor foran oligosaccharide and/or carbohydrate.

As used herein, the term “aglycosylation” or “aglycosylated” refers tothe production of a molecule in an unglycosylated form (e.g., byengineering a protein or polypeptide to lack amino acid residues thatserve as acceptors of glycosylation). Alternatively, a protein orpolypeptide can be expressed in, e.g., E. coli, to produce anaglycosylated protein or polypeptide.

As used herein, the term “deglycosylation” or “deglycosylated” refers tothe process or result of enzymatic removal of sugar moieties on amolecule.

As used herein, the term “underglycosylation” or “underglycosylated”refers to a molecule in which one or more carbohydrate structures thatwould normally be present if produced in a mammalian cell has beenomitted, removed, modified, or masked.

As used herein, the term “Fc region” and “Fc domain” is the portion of anative immunoglobulin formed by the respective Fc domains (or Fcmoieties) of its two heavy chains without the variable regions whichbind antigen. In some embodiments, an Fc domain begins in the hingeregion just upstream of the papain cleavage site and ending at theC-terminus of the antibody. Accordingly, a complete Fc domain comprisesat least a hinge domain, a CH2 domain, and a CH3 domain. In certainembodiments, an Fc domain comprises at least one of: a hinge (e.g.,upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3domain, a CH4 domain, or a variant, portion, or fragment thereof. Inother embodiments, an Fc domain comprises a complete Fc domain (i.e., ahinge domain, a CH2 domain, and a CH3 domain). In one embodiment, an Fcdomain comprises a hinge domain (or portion thereof) fused to a CH3domain (or portion thereof). In another embodiment, an Fc domaincomprises a CH2 domain (or portion thereof) fused to a CH3 domain (orportion thereof). In another embodiment, an Fc domain consists of a CH3domain or portion thereof. In another embodiment, an Fc domain consistsof a hinge domain (or portion thereof) and a CH3 domain (or portionthereof). In another embodiment, an Fc domain consists of a CH2 domain(or portion thereof) and a CH3 domain. In another embodiment, an Fcdomain consists of a hinge domain (or portion thereof) and a CH2 domain(or portion thereof). In one embodiment, an Fc domain lacks at least aportion of a CH2 domain (e.g., all or part of a CH2 domain). In oneembodiment, an Fc domain of the invention comprises at least the portionof an Fc molecule known in the art to be required for FcRn binding. Inone embodiment, an Fc domain of the invention comprises at least theportion of an Fc molecule known in the art to be required for Protein Abinding. In one embodiment, an Fc domain of the invention comprises atleast the portion of an Fc molecule known in the art to be required forprotein G binding. An Fc domain herein generally refers to a polypeptidecomprising all or part of the Fc domain of an immunoglobulinheavy-chain. This includes, but is not limited to, polypeptidescomprising the entire CHI, hinge, CH2, and/or CH3 domains as well asfragments of such peptides comprising only, e.g., the hinge, CH2, andCH3 domain. The Fc domain may be derived from an immunoglobulin of anyspecies and/or any subtype, including, but not limited to, a human IgG1,IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody. The Fc domainencompasses native Fc and Fc variant molecules. As with Fc variants andnative Fc's, the term Fc domain includes molecules in monomeric ormultimeric form, whether digested from whole antibody or produced byother means.

As set forth herein, it will be understood by one of ordinary skill inthe art that any Fc domain may be modified such that it varies in aminoacid sequence from the native Fc domain of a naturally occurringimmunoglobulin molecule.

The Fc domains of an interferon receptor Fc construct of the disclosuremay be derived from different immunoglobulin molecules. For example, anFc domain of an interferon receptor Fc construct may comprise a CH2and/or CH3 domain derived from an IgG1 molecule and a hinge regionderived from an IgG3 molecule. In another example, an Fc domain cancomprise a chimeric hinge region derived, in part, from an IgG1 moleculeand, in part, from an IgG3 molecule. In another example, an Fc domaincan comprise a chimeric hinge derived, in part, from an IgG1 moleculeand, in part, from an IgG4 molecule. The wild type human IgG1 Fc domainhas the amino acid sequence set forth in SEQ ID NO: 26.

As used herein, the term “serum half-life” refers to the time requiredfor the in vivo serum soluble interferon receptor concentration todecline by 50%. The shorter the serum half-life of the solubleinterferon receptor, the shorter time it will have to exert atherapeutic effect.

As used herein, the term “soluble interferon receptor” or “heterodimer”refers to a molecule comprising a first IFNAR domain, or variant orfragment thereof, and a second IFNAR domain, or a variant or fragmentthereof. In some embodiments, an IFNAR domain is the extracellulardomain of an IFNAR. In some embodiments, the soluble interferon receptorcomprises a first polypeptide and a second polypeptide, wherein thefirst polypeptide comprises the extracellular domain of an IFNAR, or avariant or fragment thereof, and the second polypeptide comprises theextracellular domain of an IFNAR, or a variant or fragment thereof. Insome embodiments, the first and second polypeptide interact to form adimer (e.g., a heterodimer). In some embodiments, a soluble interferonreceptor comprises a first polypeptide comprising an IFNAR domain, or avariant or fragment thereof operably linked, with or without a linkerdomain, to an immunoglobulin Fc domain, or a variant or fragmentthereof, and wherein the second polypeptide comprises an IFNAR domain,or a variant or fragment thereof operably linked, with or without alinker domain, to an immunoglobulin Fc domain, or a variant or fragmentthereof; and nucleic acids encoding such polypeptides. In someembodiments, the first polypeptide comprises INFAR1, or a variant orfragment thereof operably linked, with or without a linker domain, to aFc domain, or variant or fragment thereof. In some embodiments, thesecond polypeptide comprises INFAR2, or a variant or fragment thereofoperably linked, with or without a linker domain, to a variant Fcdomain, or variant or fragment thereof. In some embodiments, the firstand second polypeptides dimerize to form a heterodimeric construct. Insome embodiments, the first and/or second polypeptides may or may notcomprise a leader sequence. In some embodiments, the soluble interferonreceptor may or may not comprise a leader sequence. In some embodiments,the soluble interferon receptors comprise two or more interferonreceptor Fc constructs. In some embodiments, the soluble interferonreceptor binds type I interferons, e.g., IFN-α, INF-β, IFN-ε, -κ, -τ,-ζ, IFN-ω, IFN-ν.

As used herein, the term “interferon receptor Fc construct” refers to apolypeptide comprising an IFNAR domain (e.g., IFNAR1 or IFNAR2), or avariant or fragment thereof operably linked, with or without a linkerdomain, to an Fc domain, or a variant or fragment thereof, and nucleicacids encoding such polypeptides. In some embodiments, the IFNAR domainis the extracellular domain of an IFNAR. The interferon receptor Fcconstruct may or may not comprise a leader sequence. The interferonreceptor Fc construct may also be referred to as an “interferon receptorFc protein”.

As used herein, the term “type I interferon” or “type I IFN” refers to apro-inflammatory cytokine that binds to a heterodimeric cell surfacereceptor, IFN-α receptor (IFNAR), comprising IFNAR1 and IFNAR2. Thereare sixteen type I IFNs in humans, including IFN-α, INF-β, IFN-ε, -κ,-τ, -ζ, IFN-ω, IFN-ν. Type I IFNs are rapidly produced in multipledifferent cell types, and known to have a wide variety of effects.Although all type I IFNs bind the same receptor and form structurallyvery similar ternary complexes, differential IFN activities result fromdifferent lifetimes and ligand affinities on each receptor chain, whichdictate assembly and dynamics of the signaling complex (Piehler, J. etal. (2012) Immunological Reviews 250: 317-334; Li, H. et al. (2017) JMol Biol 429: 2571-2589).

As used herein, the term “type I IFN activity” refers to a biologicalactivity which results upon interaction of a type I IFN to a receptor(IFNAR), including, but not limited to, those described herein. Thecanonical consequences of type I IFN production in vivo is theactivation of antimicrobial cellular programs and the development ofinnate and adaptive immune responses. Type I IFN modulates innate immunecell activation (e.g., maturation of dendritic cells) to promote antigenpresentation and natural killer cell functions. Type I IFN also promotesthe development of high-affinity antigen-specific T and B cell responsesand immunological memory (Ivashkiv and Donlin (2014) Nat Rev Immunol14(1):36-49).

Type I IFNs have been shown to activate dendritic cells (DCs) andpromote their T cell stimulatory capacity through autocrine signaling(Montoya et al., (2002) Blood 99:3263-3271). Type I IFN exposurefacilitates maturation of DCs via increasing the expression of chemokinereceptors and adhesion molecules (e.g., to promote DC migration intodraining lymph nodes), co-stimulatory molecules, and MHC class I andclass II antigen presentation. DCs that mature following type I IFNexposure can effectively prime protective T cell responses (Wijesundaraet al., (2014) Front Immunol 29(412) and references therein).Accordingly, in some embodiments, type I IFN activity is an increase orinduction in DC activation and/or maturation. In some embodiments, typeI IFN activity is an increase or induction in DC migration to draininglymph nodes.

Further, type I IFN can either promote T cell activation, proliferation,differentiation and survival (Crouse et al., (2015) Nat Rev Immunol15:231-242). Early studies revealed that MHC-I expression is upregulatedin response to type I IFN in multiple cell types (Lindahl et al.,(1976), J Infect Dis 133(Suppl):A66-A68; Lindahl et al., (1976) ProcNatl Acad Sci USA 17:1284-1287) which is a requirement for optimal Tcell stimulation, differentiation, expansion and cytolytic activity. Inaddition, type I IFNs can exert potent co-stimulatory effects on CD8 Tcells, enhancing CD8 T cell proliferation and differentiation(Curtsinger et al., (2005) J Immunol 174:4465-4469; Kolumam et al.,(2005) J Exp Med 202:637-650). Accordingly, in some embodiments, type IIFN activity is an increase or induction in T cell proliferation. Insome embodiments, type I IFN activity is an increase or induction inCD8+ T cell proliferation. In some embodiments, type I IFN activity isan increase or induction in T cell differentiation. In some embodiments,type I IFN activity is an increase or induction in CD8+ T celldifferentiation.

With regards to B cells, type I IFN exposure has been shown to promote Bcell activation, antibody production and isotype switch following viralinfection or following experimental immunization (Le Bon et al., (2006)J Immunol 176:4:2074-2078; Swanson et al., (2010) J Exp Med207:1485-1500). Accordingly, in some embodiments, type I IFN activity isan increase or induction in antibody production.

In some embodiments, a type I IFN activity results upon IFNα binding toIFNAR. In some embodiments, a type I IFN activity results upon IFNβbinding to IFNAR. In some embodiments, a type I IFN activity is selectedfrom the group consisting of: (i) an increase or induction in T cellproliferation (e.g., CD8+ T cell proliferation); (ii) an increase orinduction in DC maturation; (iii) an increase or induction in DCmigration into draining lymph nodes; (iv) an increase or induction in Tcell differentiation (e.g., CD8+ T cell differentiation); (v) anincrease or induction in antibody production; and (vi) any combinationof (i)-(v). In some embodiments, a type I IFN activity is determined invitro. In some embodiments, a type I IFN activity is determined in vivo.

In some embodiments, the heterodimers described herein inhibit or reducea type I IFN activity. For example, in some embodiments, theheterodimers described herein: (i) inhibit or reduce T cellproliferation (e.g., CD8+ T cell proliferation); (ii) inhibitor reduceDC maturation; (iii) inhibit or reduce DC migration into draining lymphnodes; (iv) inhibit or reduce T cell differentiation (e.g., CD8+ T celldifferentiation); (v) inhibit or reduce IFN-mediated antibodyproduction; or (vi) any combination of (i)-(v). In some embodiments,inhibition or reduction of type I IFN activity by the heterodimersdescribed herein is relative to the activity in the absence of aheterodimer. Methods for determining inhibition or reduction of T cellproliferation, DC maturation, DC migration, T cell differentiation, andIFN-mediated antibody production are known to those of skill in the art.

As used herein, the term “dimer” refers to a macromolecular complexformed by two macromolecules (e.g., polypeptides). A “homodimer” refersto a dimer that is formed by two identical macromolecules (e.g.,polypeptides). A “heterodimer” refers to a dimer that is formed by twodifferent macromolecules (e.g., polypeptides).

As used herein, the term “variant” refers to a polypeptide derived froma wild-type interferon receptor or Fc domain and differs from thewild-type by one or more alteration(s), i.e., a substitution, insertion,and/or deletion, at one or more positions. A substitution means areplacement of an amino acid occupying a position with a different aminoacid. A deletion means removal of an amino acid occupying a position. Aninsertion means adding 1 or more, such as 1-3 amino acids, immediatelyadjacent to an amino acid occupying a position. Variant polypeptidesnecessarily have less than 100% sequence identity or similarity with thewild-type polypeptide. In some embodiments, the variant polypeptide willhave an amino acid sequence from about 75% to less than 100% amino acidsequence identity or similarity with the amino acid sequence ofwild-type polypeptide, or from about 80% to less than 100%, or fromabout 85% to less than 100%, or from about 90% to less than 100% (e.g.,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% o, 99%) or from about 95% to lessthan 100%, e.g., over the length of the variant polypeptide.

In certain aspects, the interferon receptor Fc constructs employ one ormore “linker domains,” such as polypeptide linkers. As used herein, theterm “linker domain” refers to one or more amino acids which connect twoor more peptide domains in a linear polypeptide sequence. As usedherein, the term “polypeptide linker” refers to a peptide or polypeptidesequence (e.g., a synthetic peptide or polypeptide sequence) whichconnects two or more polypeptide domains in a linear amino acid sequenceof a protein. For example, polypeptide linkers may be used to operablylink an interferon receptor to an Fc domain. Such polypeptide linkers insome embodiments provide flexibility to the polypeptide molecule. Insome embodiments the polypeptide linker is used to connect (e.g.,genetically fuse), for example, an IFNAR1 domain to an Fc domain and/oran IFNAR2 domain to an Fc domain. An interferon receptor Fc constructmay include more than one linker domain or peptide linker. Variouspeptide linkers are known in the art.

As used herein, the term “gly-ser polypeptide linker” refers to apeptide that consists of glycine and serine residues. An exemplarygly/ser polypeptide linker comprises the amino acid sequence (Gly₄Ser)n.In some embodiments, n is 1 or more, such as 2 or more, 3 or more, 4 ormore, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 ormore (e.g., (Gly₄Ser)10). Another exemplary gly/ser polypeptide linkercomprises the amino acid sequence Ser(Gly₄Ser)n. In some embodiments, nis 1 or more, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, or 10 or more (e.g.,Ser(Gly₄Ser)10).

As used herein, the terms “coupled,” “conjugated,” “linked,” “fused,” or“fusion,” are used interchangeably. These terms refer to the joiningtogether of two more elements or components or domains, by whatevermeans including chemical conjugation or recombinant means. Methods ofchemical conjugation (e.g., using heterobifunctional crosslinkingagents) are known in the art.

A polypeptide or amino acid sequence “derived from” a designatedpolypeptide or protein refers to the origin of the polypeptide.Preferably, the polypeptide or amino acid sequence which is derived froma particular sequence has an amino acid sequence that is essentiallyidentical to that sequence or a portion thereof, wherein the portionconsists of at least 10-20 amino acids, preferably at least 20-30 aminoacids, more preferably at least 30-50 amino acids, or which is otherwiseidentifiable to one of ordinary skill in the art as having its origin inthe sequence. Polypeptides derived from another polypeptide may have oneor more mutations relative to the starting polypeptide, e.g., one ormore amino acid residues which have been substituted with another aminoacid residue or which has one or more amino acid residue insertions ordeletions.

In one embodiment, there is one amino acid difference between a startingpolypeptide sequence and the sequence derived therefrom. Identity orsimilarity with respect to this sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical (i.e., same residue) with the starting amino acid residues,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity.

In one embodiment, a polypeptide of the disclosure consists of, consistsessentially of, or comprises an amino acid sequence as set forth in theSequence Listing or Sequence Table disclosed herein and functionallyactive variants thereof. In an embodiment, a polypeptide includes anamino acid sequence at least 80%, such as at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to an amino acid sequence set forthin the Sequence Listing or Sequence Table disclosed herein. In someembodiments, a polypeptide includes a contiguous amino acid sequence atleast 80%, such as at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to a contiguous amino acid sequence set forth in theSequence Listing or Sequence Table disclosed herein. In someembodiments, a polypeptide includes an amino acid sequence having atleast 10, such as at least 15, at least 20, at least 25, at least 30, atleast 35, at least 40, at least 45, at least 50, at least 55, at least60, at least 65, at least 70, at least 75, at least 80, at least 85, atleast 90, at least 95, at least 100, at least 200, at least 300, atleast 400, or at least 500 (or any integer within these numbers)contiguous amino acids of an amino acid sequence set forth in SequenceListing or Sequence Table disclosed herein.

In some embodiments, the interferon receptor Fc constructs of thedisclosure are encoded by a nucleotide sequence. Nucleotide sequences ofthe disclosure can be useful for a number of applications, including:cloning, gene therapy, protein expression and purification, mutationintroduction, DNA vaccination of a host in need thereof, antibodygeneration for, e.g., passive immunization, PCR, primer and probegeneration, siRNA design and generation (see, e.g., the DharmaconsiDesign website), and the like. In some embodiments, the nucleotidesequence of the disclosure comprises, consists of, or consistsessentially of, a nucleotide sequence that encodes the amino acidsequence of the interferon receptor Fc constructs selected from theSequence Table or Sequence Listing. In some embodiments, a nucleotidesequence includes a nucleotide sequence at least 80%, such as at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to anucleotide sequence encoding an amino acid sequence of the SequenceListing or Sequence Table disclosed herein. In some embodiments, anucleotide sequence includes a contiguous nucleotide sequence at least80%, such as at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to a contiguous nucleotide sequence encoding an amino acidsequence set forth in the Sequence Listing or Sequence Table disclosedherein. In some embodiments, a nucleotide sequence includes a nucleotidesequence having at least 10, such as at least 15, such as at least 20,at least 25, at least 30, at least 35, at least 40, at least 45, atleast 50, at least 55, at least 60, at least 65, at least 70, at least75, at least 80, at least 85, at least 90, at least 95, at least 100, atleast 200, at least 300, at least 400, or at least 500 (or any integerwithin these numbers) contiguous nucleotides of a nucleotide sequenceencoding an amino acid sequence set forth in the Sequence Listing orSequence Table disclosed herein.

It will also be understood by one of ordinary skill in the art that theinterferon receptor Fc constructs may be altered such that they vary insequence from the naturally occurring or native sequences from whichtheir components (e.g., interferon receptor domains, linker domains, andFc domains) are derived, while retaining the desirable activity of thenative sequences. For example, nucleotide or amino acid substitutionsleading to conservative substitutions or changes at “non-essential”amino acid residues may be made. An isolated nucleic acid moleculeencoding a non-natural variant can be created by introducing one or morenucleotide substitutions, additions or deletions into the nucleotidesequence of the interferon receptor Fc constructs such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.

The interferon receptor Fc constructs may comprise conservative aminoacid substitutions at one or more amino acid residues, e.g., atessential or non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart, including basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine), andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a nonessential amino acid residue in an interferonreceptor Fc construct is preferably replaced with another amino acidresidue from the same side chain family. In another embodiment, a stringof amino acids can be replaced with a structurally similar string thatdiffers in order and/or composition of side chain family members.Alternatively, in another embodiment, mutations may be introducedrandomly along all or part of a coding sequence, such as by saturationmutagenesis, and the resultant mutants can be incorporated into theinterferon receptor Fc constructs and screened for their ability to bindto the desired target.

The term “ameliorating” refers to any therapeutically beneficial resultin the treatment of a disease state, e.g., an autoimmune disease state(e.g., SLE, Sjogren's syndrome), including prophylaxis, lessening in theseverity or progression, remission, or cure thereof.

The term “in situ” refers to processes that occur in a living cellgrowing separate from a living organism, e.g., growing in tissueculture.

The term “in vivo” refers to processes that occur in a living organism.

The term “mammal” or “subject” or “patient” as used herein includes bothhumans and non-humans and include but is not limited to humans,non-human primates, canines, felines, murines, bovines, equines, andporcines.

The term percent “identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, thepercent “identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv Appl Math 1981;2:482, by the homology alignment algorithm of Needleman & Wunsch, J MolBiol 1970; 48:443, by the search for similarity method of Pearson &Lipman, PNAS 1988; 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by visual inspection (see generally Ausubel et al, infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J Mol Biol 1990; 215:403-10. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information website.

The term “sufficient amount” means an amount sufficient to produce adesired effect.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate a symptom of a disease. A therapeuticallyeffective amount can be a “prophylactically effective amount” asprophylaxis can be considered therapy.

The term “about” will be understood by persons of ordinary skill andwill vary to some extent depending on the context in which it is used.If there are uses of the term which are not clear to persons of ordinaryskill given the context in which it is used, “about” will mean up toplus or minus 10% of the particular value.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

Soluble Interferon Receptors

In some embodiments, the soluble interferon receptors of the disclosureinclude a first polypeptide and a second polypeptide, wherein eachpolypeptide comprises an interferon receptor Fc construct with orwithout a leader sequence. An interferon receptor Fc construct includesan interferon receptor domain (e.g., IFNAR1 or IFNAR2), with or withouta leader sequence, or a variant or fragment thereof, operably coupledwith or without a linker domain to a Fc domain, or a variant or fragmentthereof. In some embodiments, the first and second polypeptides dimerizeto form a dimer; for example, a heterodimer.

In some embodiments, a composition of the disclosure includes a solubleinterferon receptor.

In some embodiment, the interferon receptor domain is operably coupledto an Fc domain, or a variant of fragment thereof without a linkerdomain.

In some embodiments, the interferon receptor domain is operably coupledto an Fc domain, or a variant or fragment thereof, via a linker domain.In some embodiments, the linker domain is a linker peptide. In someembodiments, the linker domain is a linker nucleotide.

In some embodiments, linker domains include (gly4ser) 2, 3, 4 or 5variants that alter the length of the linker by 5 amino acidprogressions. In another embodiment, a linker domain is approximately 18amino acids in length and includes an N-linked glycosylation site, whichcan be sensitive to protease cleavage in vivo. In some embodiments, anN-linked glycosylation site can protect the soluble interferon receptorfrom cleavage in the linker domain. In some embodiments, an N-linkedglycosylation site can assist in separating the folding of independentfunctional domains separated by the linker domain.

In some embodiments, the linker domain is an NLG linker(VDGASSPVNVSSPSVQDI) (SEQ ID NO: 18).

In some embodiments, the interferon receptor Fc construct includes aleader molecule, e.g., a leader peptide. In some embodiments, the leadermolecule is a leader peptide positioned at the N-terminus of theinterferon receptor domain. In some embodiments, an interferon receptorFc construct of the invention comprises a leader peptide at theN-terminus of the molecule, wherein the leader peptide is later cleavedfrom the interferon receptor Fc construct. Methods for generatingnucleic acid sequences encoding a leader peptide fused to a recombinantprotein are well known in the art. In some embodiments, any of theinterferon receptor Fc constructs of the present invention can beexpressed or synthesized either with or without a leader fused to theirN-terminus. The protein sequence of an interferon receptor Fc constructof the present disclosure following cleavage of a fused leader peptidecan be predicted and/or deduced by one of skill in the art.

In some embodiments, the leader peptide comprises the amino acidsequence: MDWTWRILFLVAAATGTHA (SEQ ID NO: 13). In some embodiments theleader is a VK3 leader peptide (VK3LP), which comprises the amino acidsequence: METPAQLLFLLLLWLPDTTG (SEQ ID NO: 14). In some embodiments, theleader peptide is fused to the N-terminus of the interferon receptor Fcconstruct. Such leader sequences can improve the level of synthesis andsecretion of the interferon receptor Fc construct in mammalian cells. Insome embodiments, the leader is cleaved, yielding interferon receptor Fcconstructs. In some embodiments, an interferon receptor Fc construct ofthe present invention is expressed without a leader peptide fused to itsN-terminus, and the resulting interferon receptor Fc construct has anN-terminal methionine.

In some embodiments, the soluble interferon receptors of the disclosureinclude a first and a second polypeptide, wherein each of thepolypeptides includes an interferon receptor domain (e.g. IFNAR1,IFNAR2), with or without a leader sequence, or variant or fragmentthereof operably coupled, with or without a linker domain, to the N- orC-terminus of an immunoglobulin Fc domain, or a variant or fragmentthereof. In some embodiments, the interferon receptor domains of each ofthe two polypeptides are different, e.g., IFNAR1 and IFNAR2. In someembodiments, a soluble interferon receptor is a dimeric molecule. Insome embodiments, the first and second polypeptides dimerize to form aheterodimer.

In some embodiments, the interferon receptor Fc construct includessubstantially all or at least an active fragment of an interferonreceptor. In some embodiments, the interferon receptor is IFNAR1, forexample, the extracellular domain of a human IFNAR1 (SEQ ID NO: 11), ora variant or fragment thereof. In some embodiments, the interferonreceptor is IFNAR2, for example, a the extracellular domain of humanIFNAR2 (SEQ ID NO: 12), or a variant or fragment thereof. In someembodiments, a IFNAR-linker-Fc containing a 20 or 25 aa linker domain ismade. In some embodiments, a IFNAR-linker-Fc containing a 10 aa linkerdomain is made. In some embodiments, a IFNAR-linker-Fc containing a 5 aalinker domain is made. In some embodiments, a IFNAR-Fc without a linkerdomain is made. In some embodiments, the interferon receptor may or maynot include a leader sequence.

In some embodiments, interferon receptor Fc constructs includeIFNAR-linker-Fc, wherein the IFNAR domain is located at the COOH side ofthe Fc. In other embodiments, interferon receptor Fc constructs includeIFNAR-linker-Fc, wherein the IFNAR domain is located at the NH2 side ofthe Fc. In some embodiments, soluble interferon receptors include:IFNAR1-Fc and IFNAR2-Fc; IFNAR1-linker-Fc and IFNAR2-linker-Fc;Fc-IFNAR1 and Fc-IFNAR2; Fc-linker-IFNAR1 and Fc-linker-IFNAR2. Theinterferon receptor Fc constructs may or may not include a leadersequence.

In some embodiments, fusion junctions between interferon receptordomains and the other domains of the interferon receptor Fc constructsare optimized.

FIG. 1 displays exemplary configurations of the soluble interferonreceptors, and the Sequence Table provides the sequences of exemplaryinterferon receptor Fc constructs of various configurations.

In one embodiment, the interferon receptor domain is operably coupled(e.g., chemically conjugated or genetically fused (e.g., either directlyor via a linker, such as a polypeptide linker)) to the N-terminus of aFc domain, or a variant or fragment thereof. In another embodiment, theinterferon receptor domain is operably coupled (e.g., chemicallyconjugated or genetically fused (e.g., either directly or via a linker,such as a polypeptide linker)) to the C-terminus of a Fc domain, or avariant or fragment thereof. In other embodiments, an interferonreceptor domain is operably coupled (e.g., chemically conjugated orgenetically fused (e.g., either directly or via a linker, such as apolypeptide linker)) via an amino acid side chain of a Fc domain, or avariant or fragment thereof.

In some embodiments, the soluble interferon receptors comprise at leasttwo of the same or different interferon receptor domains (e.g., IFNAR1and IFNAR2), or a variant or fragment thereof, and at least two of thesame or different Fc domains, or a variant or fragment thereof, with anoptional linker domain between the interferon receptor domains and theFc domains.

In some embodiments, the interferon receptor Fc constructs form aheterodimer.

In some embodiments, the soluble interferon receptor of the disclosurecomprise a Fc domain, or a variant or fragment thereof, as describedherein, thereby increasing serum half-life and bioavailability of thesoluble interferon receptors.

It will be understood by the skilled artisan that other configurationsof the interferon receptor domains and Fc domains are possible, with theinclusion of optional linkers between the interferon receptor domain andFc domain. It will also be understood that domain orientation can bealtered, so long as the interferon receptor domains are active in theparticular configuration tested.

In certain embodiments, the soluble interferon receptor of thedisclosure have at least one interferon receptor domain specific for atarget molecule which mediates a biological effect. In anotherembodiment, binding of the soluble interferon receptor of the disclosureto a target molecule, such as type I interferon (e.g. IFN-α, INF-β,IFN-ε, -κ, -τ, -ζ, IFN-ω, IFN-ν), results in the reduction orelimination of the target molecule, e.g., from a cell, a tissue, or fromcirculation.

In other embodiments, the interferon receptor Fc constructs of thedisclosure may be assembled together or with other interferon receptorFc constructs or polypeptides to form binding proteins having two ormore polypeptides (“multimers”), wherein at least one polypeptide of themultimer is an interferon receptor Fc construct of the disclosure.Exemplary multimeric forms include dimeric, trimeric, tetrameric, andhexameric altered binding proteins and the like. In one embodiment, thepolypeptides of the multimer are the same (i.e., homomeric bindingproteins, e.g., homodimers, homotetramers). In another embodiment, thepolypeptides of the multimer are different (e.g., heteromeric bindingproteins, e.g., heterodimers).

In some embodiments, a soluble interferon receptor has a serum half-lifethat is increased at least about 1.5-fold, such as at least 3-fold, atleast 5-fold, at least 10-fold, at least about 20-fold, at least about50-fold, at least about 100-fold, at least about 200-fold, at leastabout 300-fold, at least about 400-fold, at least about 500-fold, atleast about 600-fold, at least about 700-fold, at least about 800-fold,at least about 900-fold, at least about 1000-fold, or 1000-fold orgreater relative to the corresponding interferon receptor molecules notfused to the Fc domain, or a variant or fragment thereof. In otherembodiments, a soluble interferon receptor has a serum half-life that isdecreased at least about 1.5-fold, such as at least 3-fold, at least5-fold, at least 10-fold, at least about 20-fold, at least about50-fold, at least about 100-fold, at least about 200-fold, at leastabout 300-fold, at least about 400-fold, at least about 500-fold, or500-fold or lower relative to the corresponding interferon receptormolecules not fused to the Fc domain, or a variant or fragment thereof.Routine art-recognized methods can be used to determine the serumhalf-life of the soluble interferon receptors of the disclosure.

In some embodiments, the activity of the interferon receptor (e.g.,IFNAR1 or IFNAR2) in the soluble interferon receptor is not less thanabout 10-fold less, such as 9-fold less, 8-fold less, 7-fold less,6-fold less, 5-fold less, 4-fold less, 3-fold less, or 2-fold less thanthe activity of a naturally coccuring, wild type interferon receptormolecule. In some embodiments, the activity of the interferon receptorin the soluble interferon receptor is about equal to the activity of anaturally occurring, wild type interferon receptor molecule.

In some embodiments, the soluble interferon receptor can bind to anddecrease the effects of interferon.

In some embodiments, the activity of the soluble interferon receptor isdetectable in vitro and/or in vivo. In some embodiments, the solubleinterferon receptor construct binds to a cell, a diseased cell, amalignant cell, or a cancer cell and interferes with its biologicactivity.

In another aspect, a multifunctional soluble interferon receptor isprovided that is attached to an enzyme or antibody having bindingspecificity, such as an scFv targeted to type I interferons. Forexample, type I interferons include IFN-α, INF-β, IFN-ε, -κ, -τ, -ζ,IFN-ω, IFN-ν.

In some embodiments, the targets of the IFNAR domains of the solubleinterferon receptor are primarily extracellular type I interferons. Forexample, IFN-α, INF-β, IFN-ε, -κ, -τ, -ζ, IFN-ω, IFN-ν. In someembodiments, the soluble interferon receptor is active in the acidicenvironment of the endocytic vesicles. In some embodiments, a solubleinterferon receptor, including a Fc domain, or a variant or fragmentthereof, is adapted to be active both extracellularly and in theendocytic environment. In some embodiments, the IFNAR domain of asoluble interferon receptor is not active in the cytoplasm of a cell.

In some embodiments, soluble interferon receptors include both IFNAR1and IFNAR2. In some embodiments, these soluble interferon receptorsimprove therapy of SLE because they bind interferon (IFN), e.g., IFNα orIFNβ, and negate or reduce the inflammatory effects of the cytokine.

Interferon Receptors Domains

In some embodiments, the interferon receptor Fc constructs of thepresent disclosure comprise one or more interferon receptor domains(IFNAR), or a variant or fragment thereof operably coupled with orwithout a linker domain to an immunoglobulin Fc domain, or a variant orfragment thereof. In some embodiments, the interferon receptor domain isa variant or fragment of an interferon receptor domain. In someembodiments, the interferon receptor domain comprises the extracellulardomain of the interferon receptor. For example, the extracellular domainof IFNAR1 or the extracellular domain of IFNAR2. In some embodiments,the interferon receptor domain includes a leader sequence. In someembodiments, the interferon receptor domain does not include a leadersequence.

Suitable IFNAR domains are well-known in the art and include, but arenot limited to, IFNAR1 and IFNAR2. For example, IFNAR domains arediscussed in deWeerd et al., Type I Interferon Receptors: Biochemistryand Biological Functions, J. of Biol. Chem., Vol. 282, No. 28,20053-20057 (2007), the entire contents of which is hereby incorporatedherein by reference.

The type I interferon-α/β receptor (IFNAR) is a heteromeric cell surfacereceptor comprised of multiple components, designated IFNAR1 and IFNAR2.The INFAR complex is unique among cytokine receptors as it binds toand/or mediates signaling by more than 15 different but related type Iinterferon (IFN) ligands, including, for example, IFN-α and IFN-β,several IFNα subtypes, IFN-ω, IFN-ε, IFN-κ, and others. Upon binding oftype I IFNs, IFNAR activates the JAK-STAT signaling pathway.

In some embodiments, the interferon receptor Fc constructs of thedisclosure bind interferon (e.g., IFN-α, INF-β, IFN-ε, -κ, -τ, -ζ,IFN-ω, IFN-ν) when complexed with another interferon receptor Fcconstruct in the form of a heterodimer. For example, a heterodimer ofthe disclosure capable of binding interferon comprises a firstinterferon receptor Fc construct, wherein the IFNAR is IFNAR1, and asecond interferon receptor Fc construct, wherein the IFNAR is IFNAR2.

In some embodiments, the IFNAR domain of an interferon receptor Fcconstruct is a naturally occurring wild-type IFNAR1 protein having theamino acid sequence set forth as SEQ ID NO:5. In some embodiments, theIFNAR domain is a variant or fragment of the IFNAR1 protein. In someembodiments, the IFNAR domain comprises 50-100, 100-150, 150-200,200-250, 250-300, 300-350, 350-400, 400-450, 450-500, or 450-557 aminoacids of the amino acids sequence set forth as SEQ ID NO: 5. In someembodiments, the IFNAR domain comprises 350-360, 360-370, 370-380,380-390, 390-400, 400-410, 410-420, 420-430, 430-440, 440-450, 450-470,460-470, 470-480, 480-490, or 490-500 amino acids of the amino acidsequence set forth as SEQ ID NO: 5. In some embodiments, the IFNAR1domain comprises amino acids 28-436 of the amino acid sequence set forthas SEQ ID NO: 5.

In some embodiments, the IFNAR domain comprises an amino acid sequenceat least 70% identical, such as 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or at least 99.5% identical to the aminoacid sequence set forth as SEQ ID NO: 5.

In some embodiments, the IFNAR domain of an interferon receptor Fcconstruct comprises the amino acid sequence set forth as SEQ ID NO:7. Insome embodiments, the IFNAR domain comprises the amino acid sequence setforth as SEQ ID NO:11. In some embodiments, the IFNAR domain comprisesan amino acid sequence at least 80% identical, such as 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or at least 99.5% identical to the amino acid sequenceset forth as SEQ ID NO: 7 or SEQ ID NO: 11.

In some embodiments, the IFNAR domain is a naturally occurring humanwild-type IFNAR2 protein having the amino acid sequence set forth as SEQID NO: 6. In some embodiments, the IFNAR domain is a variant or fragmentof the IFNAR2 protein. In some embodiments, the IFNAR domain comprises50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450,450-500, or 450-515 amino acids of the amino acids sequence set forth asSEQ ID NO: 6. In some embodiments, the IFNAR domain comprises 150-160,160-170, 170-180, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240,240-250, 250-260, 260-270, 270-280, 280-290, 290-300 amino acids of theamino acids sequence set forth as SEQ ID NO: 6. In some embodiments, theIFNAR2 domain comprises amino acids 27-243 of the amino acid sequenceset forth as SEQ ID NO: 6.

In some embodiments, the IFNAR domain comprises an amino acid sequenceat least 70% identical, such as 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or at least 99.5% identical to the aminoacid sequence set forth as SEQ ID NO: 6.

In some embodiments, the IFNAR domain of an interferon receptor Fcconstruct comprises the amino acid sequence set forth as SEQ ID NO:8. Insome embodiments, the IFNAR domain comprises the amino acid sequence setforth as SEQ ID NO:12. In some embodiments, the IFNAR domain comprisesan amino acid sequence at least 80% identical, such as 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or at least 99.5% identical to the amino acid sequenceset forth as SEQ ID NO: 8 or SEQ ID NO: 12.

In some embodiments, the interferon receptor domain comprises theextracellular domain of an interferon receptor. In some embodiments, theinterferon receptor domain comprises the extracellular domain of IFNAR1.In some embodiments, the interferon receptor domain comprises theextracellular domain of IFNAR2. In some embodiments, the extracellulardomain of an interferon receptor (e.g., IFNAR1 or IFNAR2) is essentiallyfree of the transmembrane and cytoplasmic domains. In some embodiments,the extracellular domain of an interferon receptor (e.g., IFNAR1 orIFNAR2) is anything less that the transmembrane and cytoplasmic domains.

In some embodiments, the extracellular domain of IFNAR1 comprises theamino acid sequence set forth as SEQ ID NO: 7 or SEQ ID NO: 11. In someembodiments, the extracellular domains of IFNAR1 comprises amino acids28-436 of the amino acid sequence set forth as SEQ ID NO: 5. In someembodiments, the extracellular domains of IFNAR1 comprises amino acids1-436 of the amino acid sequence set forth as SEQ ID NO: 5.

In some embodiments, the extracellular domain of IFNAR2 comprises theamino acid sequence set forth as SEQ ID NO: 8 or SEQ ID NO: 12. In someembodiments, the extracellular domain of IFNAR2 domain comprises aminoacids 27-243 of the amino acid sequence set forth as SEQ ID NO: 6. Insome embodiments, the extracellular domain of IFNAR2 domain comprisesamino acids 1-243 of the amino acid sequence set forth as SEQ ID NO: 6.

In some embodiments, an IFNAR domain is altered or modified, e.g., bymutation which results in an amino acid addition, deletion, orsubstitution. As used herein, the term “IFNAR domain variant” refers toan IFNAR domain having at least one amino acid modification, such as anamino acid substitution, as compared to the wild-type IFNAR, or fragmentthereof, from which the IFNAR domain is derived. For example, whereinthe IFNAR domain is derived from a human wild-type IFNAR, a variantcomprises at least one amino acid mutation (e.g., substitution) ascompared to a wild type amino acid at the corresponding position of thehuman wild-type IFNAR. For example, wherein the IFNAR domain is derivedfrom the extracellular domain of a human IFNAR, a variant comprises atleast one amino acid mutation (e.g., substitution) as compared to anamino acid at the corresponding position of the extracellular IFNAR.

In some embodiments, the IFNAR variant comprises one or more amino acidsubstitutions at an amino acid position(s) located in the extracellulardomain of the IFNAR.

In some embodiments of the disclosure, the soluble interferon receptorsinclude one or more IFNAR domains. In some embodiments, the solubleinterferon receptors include two IFNAR domains. In some embodiments, theIFNAR domains of the soluble interferon receptors are different (e.g.,IFNAR1 and IFNAR2).

In some embodiments, the soluble interferon receptor includes a firstpolypeptide and a second polypeptide. In some embodiments, both thefirst polypeptide and the second polypeptide comprise an interferonreceptor Fc construct. In some embodiments, the IFNAR domain of theinterferon receptor Fc construct is IFNAR1, or a variant or fragmentthereof. In some embodiments, the IFNAR domain of the interferonreceptor Fc construct is IFNAR2, or a variant or fragment thereof. Insome embodiments, the IFNAR domain of the first polypeptide of thesoluble interferon receptor comprises IFNAR1, or a variant or fragmentthereof. In some embodiments, the IFNAR domain of the second polypeptideof the soluble interferon receptor comprises IFNAR2, or a variant orfragment thereof. In some embodiments, the IFNAR domain of theinterferon receptor Fc construct is human IFNAR1 or human IFNAR2. Insome embodiments, the IFNAR domain of the interferon receptor Fcconstruct is a fragment or variant of IFNAR1 or a fragment or variant ofIFNAR2. In some embodiments, the IFNAR domain of the interferon receptorFc construct is the extracellular domain of human IFNAR1 or theextracellular domain of human IFNAR2.

Linker Domains

In some embodiments, an interferon receptor Fc construct includes alinker domain. In some embodiments, an interferon receptor Fc constructincludes a plurality of linker domains. In some embodiments, the linkerdomain is a polypeptide linker. In certain aspects, it is desirable toemploy a polypeptide linker to fuse Fc, or a variant or fragmentthereof, with one or more interferon receptor domains to form aninterferon receptor Fc construct. In some embodiments, an interferonreceptor Fc construct does not include a linker domain.

In one embodiment, the polypeptide linker is synthetic. As used herein,the term “synthetic” with respect to a polypeptide linker includespeptides (or polypeptides) which comprise an amino acid sequence (whichmay or may not be naturally occurring) that is linked in a linearsequence of amino acids to a sequence (which may or may not be naturallyoccurring) (e.g., a Fc sequence) to which it is not naturally linked innature. For example, the polypeptide linker may comprise non-naturallyoccurring polypeptides which are modified forms of naturally occurringpolypeptides (e.g., comprising a mutation such as an addition,substitution or deletion) or which comprise a first amino acid sequence(which may or may not be naturally occurring). The polypeptide linkersof the invention may be employed, for instance, to ensure that Fc, or avariant or fragment thereof, is juxtaposed to ensure proper folding andformation of a functional Fc, or a variant or fragment thereof.Preferably, a polypeptide linker compatible with the instant inventionwill be relatively non-immunogenic and not inhibit any non-covalentassociation among monomer subunits of a binding protein.

In certain embodiments, the interferon receptor Fc construct employs anNLG linker as set forth in SEQ ID NO: 18.

In certain embodiments, the interferon receptor Fc constructs of thedisclosure employ a polypeptide linker to join any two or more domainsin frame in a single polypeptide chain. In one embodiment, the two ormore domains may be independently selected from any of the Fc domains,or variants or fragments thereof, or interferon receptor domainsdiscussed herein. For example, in certain embodiments, a polypeptidelinker can be used to fuse an Fc domain, or variant or fragment thereofto an IFNAR. In some embodiments, a polypeptide linker of the inventioncan be used to genetically fuse the C-terminus of an IFNAR to theN-terminus of a Fc domain, or variant or fragment thereof to form aninterferon receptor Fc construct. In other embodiments, a polypeptidelinker of the invention can be used to genetically fuse the C-terminusof a Fc domain, or variant or fragment thereof to the N-terminus of anIFNAR to form and interferon receptor Fc construct. In otherembodiments, a polypeptide linker of the invention can be used togenetically fuse the C-terminus of an IFNAR to the C-terminus of a Fcdomain, or variant or fragment thereof to form and interferon receptorFc construct. In other embodiments, a polypeptide linker of theinvention can be used to genetically fuse the N-terminus of an IFNAR tothe N-terminus of a Fc domain, or variant or fragment thereof to formand interferon receptor Fc construct.

In one embodiment, a polypeptide linker comprises a portion of a Fcdomain, or a variant or fragment thereof. For example, in oneembodiment, a polypeptide linker can comprise a Fc fragment (e.g., C orN domain), or a different portion of a Fc domain or variant thereof.

In another embodiment, a polypeptide linker comprises or consists of agly-ser linker. As used herein, the term “gly-ser linker” refers to apeptide that consists of glycine and serine residues. An exemplarygly/ser linker comprises an amino acid sequence of the formula(Gly₄Ser)n, wherein n is a positive integer (e.g., 1-10, 1, 2, 3, 4, or5). A preferred gly/ser linker is (Gly₄Ser)₁. Another preferred gly/serlinker is (Gly₄Ser)₂. Another preferred gly/ser linker is (Gly₄Ser)₃.Another preferred gly/ser linker is (Gly₄Ser)₄. Another preferredgly/ser linker is (Gly₄Ser)₅. In certain embodiments, the gly-ser linkermay be inserted between two other sequences of the polypeptide linker(e.g., any of the polypeptide linker sequences described herein). Inother embodiments, a gly-ser linker is attached at one or both ends ofanother sequence of the polypeptide linker (e.g., any of the polypeptidelinker sequences described herein). In yet other embodiments, two ormore gly-ser linker are incorporated in series in a polypeptide linker.

In other embodiments, a polypeptide linker of the invention comprises abiologically relevant peptide sequence or a sequence portion thereof.For example, a biologically relevant peptide sequence may include, butis not limited to, sequences derived from an anti-rejection oranti-inflammatory peptide. Said anti-rejection or anti-inflammatorypeptides may be selected from the group consisting of a cytokineinhibitory peptide, a cell adhesion inhibitory peptide, a thrombininhibitory peptide, and a platelet inhibitory peptide. In a preferredembodiment, a polypeptide linker comprises a peptide sequence selectedfrom the group consisting of an IL-1 inhibitory or antagonist peptidesequence, an erythropoietin (EPO)-mimetic peptide sequence, athrombopoietin (TPO)-mimetic peptide sequence, G-CSF mimetic peptidesequence, a TNF-antagonist peptide sequence, an integrin-binding peptidesequence, a selectin antagonist peptide sequence, an anti-pathogenicpeptide sequence, a vasoactive intestinal peptide (VIP) mimetic peptidesequence, a calmodulin antagonist peptide sequence, a mast cellantagonist, a SH3 antagonist peptide sequence, an urokinase receptor(UKR) antagonist peptide sequence, a somatostatin or cortistatin mimeticpeptide sequence, and a macrophage and/or T-cell inhibiting peptidesequence. Exemplary peptide sequences, any one of which may be employedas a polypeptide linker, are disclosed in U.S. Pat. No. 6,660,843, whichis incorporated by reference herein.

Other linkers that are suitable for use in interferon receptor Fcconstructs are known in the art, for example, the serine-rich linkersdisclosed in U.S. Pat. No. 5,525,491, the helix forming peptide linkers(e.g., A(EAAAK)nA (n=2-5)) disclosed in Arai et al., Protein Eng 2001;14:529-32, and the stable linkers disclosed in Chen et al., Mol Pharm2011; 8:457-65, i.e., the dipeptide linker LE, a thrombin-sensitivedisulfide cyclopeptide linker, and the alpha-helix forming linkerLEA(EAAAK)₄ALEA(EAAAK)₄ALE (SEQ ID NO: 19).

Other exemplary linkers include GS linkers (i.e., (GS)n), GGSG (SEQ IDNO: 27) linkers (i.e., (GGSG)n), GSAT linkers (SEQ ID NO: 28), SEGlinkers, and GGS linkers (i.e., (GGSGGS)n), wherein n is a positiveinteger (e.g., 1, 2, 3, 4, or 5). Other suitable linkers for use in theinterferon receptor Fc constructs can be found using publicly availabledatabases, such as the Linker Database (ibi.vu.nl/programs/linkerdbwww).The Linker Database is a database of inter-domain linkers inmulti-functional enzymes which serve as potential linkers in novelfusion proteins (see, e.g., George et al., Protein Engineering 2002;15:871-9).

It will be understood that variant forms of these exemplary polypeptidelinkers can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequenceencoding a polypeptide linker such that one or more amino acidsubstitutions, additions or deletions are introduced into thepolypeptide linker. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.

Polypeptide linkers of the disclosure are at least one amino acid inlength and can be of varying lengths. In one embodiment, a polypeptidelinker of the invention is from about 1 to about 50 amino acids inlength. As used in this context, the term “about” indicates +/−two aminoacid residues. Since linker length must be a positive integer, thelength of from about 1 to about 50 amino acids in length, means a lengthof from 1 to 48-52 amino acids in length. In another embodiment, apolypeptide linker of the disclosure is from about 5-10 amino acids inlength. In another embodiment, a polypeptide linker of the disclosure isfrom about 10-20 amino acids in length. In another embodiment, apolypeptide linker of the disclosure is from about 15-30 amino acids inlength. In another embodiment, a polypeptide linker of the disclosure isfrom about 15 to about 50 amino acids in length.

In another embodiment, a polypeptide linker of the disclosure is fromabout 20 to about 45 amino acids in length. In another embodiment, apolypeptide linker of the disclosure is from about 15 to about 25 aminoacids in length. In another embodiment, a polypeptide linker of thedisclosure is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, or 61 or more amino acids in length.

Polypeptide linkers can be introduced into polypeptide sequences usingtechniques known in the art. Modifications can be confirmed by DNAsequence analysis. Plasmid DNA can be used to transform host cells forstable production of the polypeptides produced.

Fc Domains

In some embodiments, the polypeptide comprising one or more interferonreceptor domains, or a variant or fragment thereof is operably coupled,with or without a linker domain, to a Fc domain, which serves as ascaffold as well as a means to increase the serum half-life of thepolypeptide. In some embodiments, the one or more interferon receptordomains and/or the Fc domain is aglycosylated, deglycosylated, orunderglycosylated. In some embodiments, the Fc domain is a mutant orvariant Fc domain, or a fragment of an Fc domain.

Suitable Fc domains are well-known in the art and include, but are notlimited to, Fc and Fc variants, such as those disclosed inWO2011/053982, WO 02/060955, WO 02/096948, WO05/047327, WO05/018572, andUS 2007/0111281 (the contents of the foregoing are incorporated hereinby reference). It is within the abilities of the skilled artisan to useroutine methods to introduce Fc domains (e.g., cloning, conjugation)into the interferon receptor Fc constructs disclosed herein (with orwithout altered glycosylation).

In some embodiments, the Fc domain is a wild type human IgG1 Fc, such asis shown in SEQ ID NO: 26. In some embodiments, the Fc domain is a wildtype human IgG4 Fc, such as is shown in SEQ ID NO: 112.

In some embodiments, an Fc domain is altered or modified, e.g., bymutation which results in an amino acid addition, deletion, orsubstitution. As used herein, the term “Fc domain variant” refers to anFc domain having at least one amino acid modification, such as an aminoacid substitution, as compared to the wild-type Fc from which the Fcdomain is derived. For example, wherein the Fc domain is derived from ahuman IgG1 antibody, a variant comprises at least one amino acidmutation (e.g., substitution) as compared to a wild type amino acid atthe corresponding position of the human IgG1 Fc region. The amino acidsubstitution(s) of an Fc variant may be located at a position within theFc domain referred to as corresponding to the position number that thatresidue would be given in an Fc region in an antibody (numberingaccording to EU index).

In one embodiment, the Fc variant comprises one or more amino acidsubstitutions at an amino acid position(s) located in a hinge region orportion thereof. In another embodiment, the Fc variant comprises one ormore amino acid substitutions at an amino acid position(s) located in aCH2 domain or portion thereof. In another embodiment, the Fc variantcomprises one or more amino acid substitutions at an amino acidposition(s) located in a CH3 domain or portion thereof. In anotherembodiment, the Fc variant comprises one or more amino acidsubstitutions at an amino acid position(s) located in a CH4 domain orportion thereof.

In some embodiments, the Fc variant comprises one or more of thefollowing amino acid substitutions: T350V, L351Y, F405A, and Y407V. Insome embodiments, the Fc variant comprises one or more of the followingamino acid substitutions: T350V, T366L, K392L, and T394W. In someembodiments, the Fc region has a mutation at N83 (i.e., N297 by Kabatnumbering), yielding an aglycosylated Fc region (e.g., Fc N83S).

In some embodiments, the Fc domain includes mutations in one or more ofthe three hinge region cysteines (residues 220, 226, and 229, numberingaccording to the EU index). In some embodiments, one or more of thethree hinge cysteines in the Fc domain can be mutated to SCC (SEQ ID NO:20) or SSS (SEQ ID NO: 21), where in “S” represents an amino acidsubstitution of cysteine with serine (wherein CCC refers to the threecysteines present in the wild type hinge domain). Accordingly “SCC”indicates an amino acid substitution to serine of only the firstcysteine of the three hinge region cysteines (residues 220, 226, and229, numbering according to the EU index), whereas “SSS” indicates thatall three cysteines in the hinge region are substituted with serine(residues 220, 226, and 229, numbering according to the EU index).

In some aspects, the Fc domain is a mutant human IgG1 Fc domain.

In some aspects, a mutant Fc domain comprises one or more mutations inthe hinge, CH2, and/or CH3 domains.

In some aspects, the Fc domain is a mutant human IgG4 Fc domain. In someembodiments, mutations in the IgG4 Fc domain include one or moremutations selected from the following group of mutations: F296Y, E356K,R409K, and H345R. In some embodiments, mutations in the IgG4 Fc domainincludes one or more mutation selected from the following group ofmutations: F296Y, R409K, and K439E. In some embodiments, the solubleinterferon receptors disclosed herein include a first polypeptidecomprising a mutant IgG4 Fc domain, wherein the Fc domain includesmutations F296Y, E356K, R409K, and H345R, and a second polypeptidecomprising a mutant IgG4 Fc domain, wherein the CH3 domain includesmutations F296Y, R409K, and K439E. In some embodiments, a mutant IgG4 Fcdomain comprises one or more mutations in the hinge, CH2, and/or CH3domains.

A. CH2 Substitutions

In some aspects, a mutant Fc domain includes a P238S mutation. In someaspects, a mutant Fc domain includes a P331S mutation. In some aspects,a mutant Fc domain includes a P238S mutation and a P331S mutation. Insome aspects, a mutant Fc domain comprises P238S and/or P331S, and mayinclude mutations in one or more of the three hinge cysteines (residues220, 226, and 229), numbering according to the EU index. In someaspects, a mutant Fc domain comprises P238S and/or P331S, and/or one ormore mutations in the three hinge cysteines (residues 220, 226, and229), numbering according to the EU index. In some aspects, a mutant Fcdomain comprises P238S and/or P331S, and/or mutations in a hingecysteine to SCC or in the three hinge cysteines to SSS. In some aspects,a mutant Fc domain comprises P238S and P331S and mutations in at leastone of the three hinge cysteines. In some aspects, a mutant Fc domaincomprises P238S and P331S and SCC. In some aspects, a mutant Fc domaincomprises P238S and P331S and SSS. In some aspects, a mutant Fc domainincludes P238S and SCC or SSS. In some aspects, a mutant Fc domainincludes P331S and SCC or SSS. (All numbering according to the EUindex).

In some aspects, a mutant Fc domain includes a mutation at a site ofN-linked glycosylation, such as N297, e.g., a substitution of asparaginefor another amino acid such as serine, e.g., N297S. In some aspects, amutant Fc domain includes a mutation at a site of N-linkedglycosylation, such as N297, e.g., a substitution of asparagine foranother amino acid such as serine, e.g., N297S and a mutation in one ormore of the three hinge cysteines. In some aspects, a mutant Fc domainincludes a mutation at a site of N-linked glycosylation, such as N297,e.g., a substitution of asparagine for another amino acid such asserine, e.g., N297S and mutations in one of the three hinge cysteines toSCC or all three cysteines to SSS. In some aspects, a mutant Fc domainincludes a mutation at a site of N-linked glycosylation, such as N297,e.g., a substitution of asparagine for another amino acid such asserine, e.g., N297 and one or more mutations in the CH2 domain whichdecrease FcγR binding and/or complement activation, such as mutations atP238 or P331 or both, e.g., P238S or P331S or both P238S and P331S. Insome aspects, such mutant Fc domains can further include a mutation inthe hinge region, e.g., SCC or SSS. (All numbering according to the EUindex.) In some aspects, the mutant Fc domain is as shown in theSequence Table or Sequence Listing herein.

B. CH3 Substitutions

In some embodiments, heterodimers are formed by mutations in the CH3domain of the Fc domain on the interferon receptor Fc constructsdisclosed herein. Heavy chains were first engineered forheterodimerization using a “knobs-into-holes” strategy (Rigway B, etal., Protein Eng., 9 (1996) pp. 617-621), incorporated herein byreference. The term “knob-into-hole” refers to the technology directingthe pairing of two polypeptides together in vitro or in vivo byintroducing a pertuberance (knob) into one polypeptide and a cavity(hole) into the other polypeptide at an interface in which theyinteract. See e.g., WO 96/027011, WO 98/050431, U.S. Pat. No. 5,731,168,US2007/0178552, WO2009089004, US 20090182127. In particular, acombination of mutations in the CH3 domain can be used to formheterodimers, for example, S354C, T366W in the “knob” heavy chain, andY349C, T366S, L368A, Y407V in the “hole” heavy chain. In anotherexample, T366Y in the “knob” heavy chain, and Y407T in the “hole” heavychain. In some embodiments, the soluble interferon receptors disclosedherein includes a first CH3 domain having the knob mutation T366W and asecond CH3 domain having the hole mutations T366S, L368A, and Y407V.(Numbering according to the EU index.) In some embodiments, the solubleinterferon receptors disclosed herein includes a first CH3 domain havingthe knob mutation T366Y and a second CH3 domain having the hole mutationY407T.

In some embodiments, the CH3 mutations are those described in US2012/0149876 A1, US 2017/0158779, U.S. Pat. Nos. 9,574,010, and9,562,109, each of which is incorporated herein by reference in itsentirety; and Von Kreudenstein, T. S. et al. mABs, 5 (2013). pp.646-654. incorporated herein by reference, and include the followingmutations: T350V, L351Y, F405A, and Y407V (first CH3 domain); and T350V,T366L, K392L, T394W (second CH3 domain). In some embodiments, thesoluble interferon receptors disclosed herein include a first CH3 domainhaving T350V, L351Y, F405A, and Y407V mutations and a second CH3 domainhaving T350V, T366L, K392L, T394W mutations. (Numbering according to theEU index.)

In some embodiments, heterodimers are formed by mutations in the CH3domain of the Fc domain on the interferon receptor Fc constructsdisclosed herein. In particular, a combination of mutations in the CH3domain can be used to form heterodimers with high heterodimericstability and purity; for example, See e.g., Von Kreudenstein et al.,mAbs 5:5, 646-654; September-October 2013, and US 2012/0149876 A1, US2017/0158779, U.S. Pat. Nos. 9,574,010, and 9,562,109, each of which isincorporated herein by reference in its entirety. In some embodiments,mutations in the Fc domain include one or more mutations selected fromthe following group of mutations: T350V, L351Y, F405A, and Y407V. Insome embodiments, mutations in the Fc domain include one or moremutation selected from the following group of mutations: T350V, T366L,K392L, and T394W. In some embodiments, the interferon receptor Fcconstructs disclosed herein include a CH3 domain having mutations T350V,L351Y, F405A, and Y407V. In some embodiments, the interferon receptor Fcconstructs disclosed herein include a CH3 domain having mutations T350V,T366L, K392L, and T394W. In some embodiments, the soluble interferonreceptors disclosed herein include a first polypeptide comprising amutant Fc domain, wherein the CH3 domain includes mutations T350V,L351Y, F405A, and Y407V, and a second polypeptide comprising a mutant Fcdomain, wherein the CH3 domain includes mutations T350V, T366L, K392L,and T394W.

Other mutations in the CH3 domain of the Fc domain are contemplated topreferentially form heterodimers. For example, See e.g., VonKreudenstein et al., mAbs 5:5, 646-654; September-October 2013,incorporated herein by reference). In some embodiments, mutations in theFc domain of the first polypeptide include one or more mutationsselected from, the following group of mutations: T350V, L351Y, F405A,and Y407V, and mutations in the Fc domain of the second polypeptideinclude one or more mutations selected from the following group ofmutations: T350V, T366L, K392M, and T394W. In some embodiments,mutations in the Fc domain of the first polypeptide include one or moremutations selected from the following group of mutations: L351Y, F405A,and Y407V, mutations in the Fc domain of the second polypeptide includeone or more mutations selected from the following group of mutations:T366L, K392M, and T394W.

In some embodiments, the CH3 mutations are those described by Moore. G.L. et al. (mABs, 3 (2011), pp. 546-557) and include the followingmutations: S364H and F405A (first CH3 domain); and Y349T and T394F(second CH3 domain). In some embodiments, the interferon receptor Fcconstructs disclosed herein include a first CH3 domain having S364H andF405A mutations and a second CH3 domain having Y349T and T394Fmutations. (Numbering according to the EU index.)

In some embodiments, the CH3 mutations are those described byGunasekaran. K. et at. (J. Biol. Chem., 285 (2010). pp. 19637-1%46) andinclude the following mutations: K409D and K392D (first CH3 domain); andD399K and E365K (second CH3 domain). In some embodiments, the interferonreceptor Fc constructs disclosed herein includes a first CH3 domainhaving K409D and K392D mutations and a second CH3 domain having D399Kand E365K mutations. (Numbering according to the EU index.)

The interferon receptor Fc constructs of the disclosure may employart-recognized Fc variants which are known to impart an alteration ineffector function and/or FcR binding. For example, a change (e.g., asubstitution) at one or more of the amino acid positions disclosed inInternational PCT Publications WO88/07089A1, WO96/14339A1, WO98/05787A1,WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1,WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2,WO04/029207A2, WO04/035752A2, WO04/063351 A2, WO04/074455A2,WO04/099249A2, WO05/040217A2, WO04/044859, WO05/070963A1, WO05/077981A2,WO05/092925A2, WO05/123780A2, WO06/019447A1, WO06/047350A2, andWO06/085967A2; US Patent Publication Nos. US2007/0231329,US2007/0231329, US2007/0237765, US2007/0237766, US2007/0237767,US2007/0243188, US20070248603, US20070286859, US20080057056; or U.S.Pat. Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871;6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124;6,737,056; 6,821,505; 6,998,253; 7,083,784; and 7,317,091, each of whichis incorporated by reference herein. In one embodiment, the specificchange (e.g., the specific substitution of one or more amino acidsdisclosed in the art) may be made at one or more of the disclosed aminoacid positions. In another embodiment, a different change at one or moreof the disclosed amino acid positions (e.g., the different substitutionof one or more amino acid position disclosed in the art) may be made.

Other amino acid mutations in the Fc domain are contemplated to reducebinding to the Fc gamma receptor and Fc gamma receptor subtypes. Theassignment of amino acids residue numbers to an Fc domain is inaccordance with the definitions of Kabat. See, e.g., Sequences ofProteins of Immunological Interest (Table of Contents, Introduction andConstant Region Sequences sections), 5th edition, Bethesda, Md.:NIH vol.1:647-723 (1991); Kabat et al., “Introduction” Sequences of Proteins ofImmunological Interest, US Dept of Health and Human Services, NIH, 5thedition, Bethesda, Md. vol. 1:xiii-xcvi (1991); Chothia & Lesk, J. Mol.Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989),each of which is herein incorporated by reference for all purposes.”

For example, mutations at positions 238, 239, 248, 249, 252, 254, 255,256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289,290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 322,324, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 356, 360, 373,376, 378, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437,438 or 439 of the Fc region can alter binding as described in U.S. Pat.No. 6,737,056, issued May 18, 2004, incorporated herein by reference inits entirety. This patent reported that changing Pro331 in IgG3 to Serresulted in six fold lower affinity as compared to unmutated IgG3,indicating the involvement of Pro331 in Fc gamma RI binding. Inaddition, amino acid modifications at positions 234, 235, 236, and 237,297, 318, 320 and 322 are disclosed as potentially altering receptorbinding affinity in U.S. Pat. No. 5,624,821, issued Apr. 29, 1997 andincorporated herein by reference in its entirety. (Numbering accordingto the EU index.)

Further mutations contemplated for use include, e.g., those described inU.S. Pat. App. Pub. No. 2006/0235208, published Oct. 19, 2006 andincorporated herein by reference in its entirety. This publicationsdescribe Fc variants that exhibit reduced binding to Fc gamma receptors,reduced antibody dependent cell-mediated cytotoxicity, or reducedcomplement dependent cytotoxicity, that comprise at least one amino acidmodification in the Fc region, including 232G, 234G, 234H, 235D, 235G,235H, 236I, 236N, 236P, 236R, 237K, 237L, 237N, 237P, 238K, 239R, 265G,267R, 269R, 270H, 297S, 299A, 299I, 299V, 325A, 325L, 327R, 328R, 329K,330I, 330L, 330N, 330P, 330R, and 331L (numbering is according to the EUindex), as well as double mutants 236R/237K, 236R/325L, 236R/328R,237K/325L, 237K/328R, 325L/328R, 235G/236R, 267R/269R, 234G/235G,236R/237K/325L, 236R/325L/328R, 235G/236R/237K, and 237K/325L/328R.Other mutations contemplated for use as described in this publicationinclude 227G, 234D, 234E, 234G, 234I, 234Y, 235D, 235I, 235S, 236S,239D, 246H, 255Y, 258H, 260H, 264I, 267D, 267E, 268D, 268E, 272H, 272I,272R, 281D, 282G, 283H, 284E, 293R, 295E, 304T, 324G, 324I, 327D, 327A,328A, 328D, 328E, 328F, 328I, 328M, 328N, 328Q, 328T, 328V, 328Y, 330I,330L, 330Y, 332D, 332E, 335D, an insertion of G between positions 235and 236, an insertion of A between positions 235 and 236, an insertionof S between positions 235 and 236, an insertion of T between positions235 and 236, an insertion of N between positions 235 and 236, aninsertion of D between positions 235 and 236, an insertion of V betweenpositions 235 and 236, an insertion of L between positions 235 and 236,an insertion of G between positions 235 and 236, an insertion of Abetween positions 235 and 236, an insertion of S between positions 235and 236, an insertion of T between positions 235 and 236, an insertionof N between positions 235 and 236, an insertion of D between positions235 and 236, an insertion of V between positions 235 and 236, aninsertion of L between positions 235 and 236, an insertion of G betweenpositions 297 and 298, an insertion of A between positions 297 and 298,an insertion of S between positions 297 and 298, an insertion of Dbetween positions 297 and 298, an insertion of G between positions 326and 327, an insertion of A between positions 326 and 327, an insertionof T between positions 326 and 327, an insertion of D between positions326 and 327, and an insertion of E between positions 326 and 327(numbering is according to the EU index). Additionally, mutationsdescribed in U.S. Pat. App. Pub. No. 2006/0235208 include 227G/332E,234D/332E, 234E/332E, 234Y/332E, 234I/332E, 234G/332E, 235I/332E,235S/332E, 235D/332E, 235E/332E, 236S/332E, 236A/332E, 236S/332D,236A/332D, 239D/268E, 246H/332E, 255Y/332E, 258H/332E, 260H/332E,264I/332E, 267E/332E, 267D/332E, 268D/332D, 268E/332D, 268E/332E,268D/332E, 268E/330Y, 268D/330Y, 272R/332E, 272H/332E, 283H/332E,284E/332E, 293R/332E, 295E/332E, 304T/332E, 324I/332E, 324G/332E,324I/332D, 324G/332D, 327D/332E, 328A/332E, 328T/332E, 328V/332E,328I/332E, 328F/332E, 328Y/332E, 328M/332E, 328D/332E, 328E/332E,328N/332E, 328Q/332E, 328A/332D, 328T/332D, 328V/332D, 328I/332D,328F/332D, 328Y/332D, 328M/332D, 328D/332D, 328E/332D, 328N/332D,328Q/332D, 330L/332E, 330Y/332E, 330I/332E, 332D/330Y, 335D/332E,239D/332E, 239D/332E/330Y, 239D/332E/330L, 239D/332E/330I,239D/332E/268E, 239D/332E/268D, 239D/332E/327D, 239D/332E/284E,239D/268E/330Y, 239D/332E/268E/330Y, 239D/332E/327A,239D/332E/268E/327A, 239D/332E/330Y/327A, 332E/330Y/268 E/327A,239D/332E/268E/330Y/327A, Insert G>297-298/332E, Insert A>297-298/332E,Insert S>297-298/332E, Insert D>297-298/332E, Insert G>326-327/332E,Insert A>326-327/332E, Insert T>326-327/332E, Insert D>326-327/332E,Insert E>326-327/332E, Insert G>235-236/332E, Insert A>235-236/332E,Insert S>235-236/332E, Insert T>235-236/332E, Insert N>235-236/332E,Insert D>235-236/332E, Insert V>235-236/332E, Insert L>235-236/332E,Insert G>235-236/332D, Insert A>235-236/332D, Insert S>235-236/332D,Insert T>235-236/332D, Insert N>235-236/332D, Insert D>235-236/332D,Insert V>235-236/332D, and Insert L>235-236/332D (numbering according tothe EU index) are contemplated for use. The mutant L234A/L235A isdescribed, e.g., in U.S. Pat. App. Pub. No. 2003/0108548, published Jun.12, 2003 and incorporated herein by reference in its entirety. Inembodiments, the described modifications are included eitherindividually or in combination. (Numbering according to the EU index.)

C. Alternative Scaffolds

The modular architecture of immunoglobulins can be utilized to createalternative scaffolds. For example, an alternative scaffold may be analternative Fc format. In some embodiments, an alternative scaffold maybe utilized to generate a heterodimeric construct.

In some embodiments of the present disclosure, the soluble interferonreceptor includes an alternative Fc domain. In some embodiments, analternative Fc domain is any of the suitable alternative Fc formatsknown in the art. For example the Fc domain may comprises any of thealternative Fc formats discussed in Spiess et al. Molecular Immunology,2015, October; 67 (2 Pt A)95-106, the entire contents of which isincorporated herein by reference. For example the alternative Fc formatmay be selected from any one of the following formats: (i) bispecificIgG (BsIgG), (ii) appended IgG, (iii) bispecific fragments, (iv)bispecific fusion proteins, (v) bispecific conjugates, (vi) and IgG4 Fabarm exchange, and (vii) alternative scaffolds.

In some embodiments, the heterodimers of the disclosure can be formed byutilizing alternative Fc formats. In some embodiments, the Fc domain canbe engineered to facilitate heterodimerization. In some embodiments,heterodimers are formed by mutations in the CH3 domain of the Fc domainof the soluble interferon receptors disclosed herein.

(i) Bispecific IgG (BsIgG)

Bispecific IgG is an alternative Fc format that can be utilized tofacilitate heterodimerization of two Fc domains and overcomehomodimerization. In some embodiments, the CH3 domain of an Fc can bemutated to facilitate heterodimerization.

In some embodiments, “knobs-into-holes” can be used to facilitateheterodimerization of Fc domains. A combination of mutations in the CH3domain can be used to form heterodimers. In some embodiments, the“knobs” are created by replacing small amino acid side chains at theinterface between CH3 domains with larger amino acid side chains, and“holes” are created by replacing large amino acid side chains at theinterface between CH3 domains with small amino acid side chains. In someembodiments, the soluble interferon receptors of the disclosure includea first interferon receptor Fc construct comprising a first CH3 domainhaving the knob mutation T366W and a second interferon receptor Fcconstruct comprising a second CH3 domain having the hole mutationsT366S, L368A, and Y407V. (Ridgeway et al., (1996) Prot. Eng. 9, 617-621and Atwell et al., (1997) J. Mol. Biol. 270, 26-35, both of which areincorporated herein by reference). In some embodiments, the solubleinterferon receptors of the disclosure include a first interferonreceptor Fc construct comprising a first CH3 domain having the knobmutation T366Y and a second interferon receptor Fc construct comprisinga second CH3 domain having the hole mutation Y407V (Ridgeway et al.,(1996) Prot. Eng. 9, 617-621). The “hole” mutations provide efficientpairing with the “knob” mutation to promote heterodimerization of the Fcdomains.

In certain embodiments, a “duobody” can also be used to facilitateheterodimerization of Fc domains (Labrijn et al., (2013) Proc. Natl.Acad. Sci. U.S.A. 110, 5145-5150, the entire contents of which is hereinincorporated by reference). For example, the Fc domain of a firstinterferon receptor Fc construct may include a F405L mutation and the Fcdomain of a second interferon receptor Fc construct may include a K409Rmutation.

In other embodiments, “azymetric mutations” can be introduced intointerferon receptor Fc constructs to promote the formation ofheterodimers. (Von Kreudenstein et al., (2013) mAbs 5, 646-654, theentire contents of which is incorporated herein by reference, and US2012/0149876 A1, US 2017/0158779, U.S. Pat. Nos. 9,574,010, and9,562,109, each of which is incorporated herein by reference in itsentirety). In some embodiments, the azymetric mutations include T350V,L351Y, F405A, and Y407V in the Fc domain of a first interferon receptorFc construct, and T350V, T366L, K392L, and T394W in the Fc domain of asecond interferon receptor Fc construct.

In some embodiments, “charge pair” mutations, which were identified byrational design of electrostatic steering mutations, can also beintroduced into interferon receptor Fc constructs to promoteheterodimerization (Gunasekaran et al., (2010) J. Biol. Chem. 285,19637-19646, and Strop et al., (2012) J. Mol. Biol. 420, 204-219, bothof which are incorporated herein by reference). The “charge pair”mutations create altered charge polarity across the Fc dimer interfacesuch that co-expression of electrostatically matched Fc chains supportfavorable attractive interactions thereby promoting Fc heterodimerformation. Unfavorable repulsive charge interactions suppress homodimerformation (Gunasekaran et al., (2010)). For example, in someembodiments, the Fc domain of a first interferon receptor Fc constructmay include K409D and K392D, and the Fc domain of a second interferonreceptor Fc construct may include D399K and E356K. In other embodiments,the Fc domain of a first interferon receptor Fc construct may includeD221E, P228E, L368E, and the Fc domain of a second interferon receptorFc construct may include D221R, P228R, and K409R.

Heterodimerization of the Fc domains of at least two interferon receptorFc constructs can also be facilitated by the introduction of “HA-TF”mutations. (Moore et al., (2011) mAbs 3, 546-557, the entire contents ofwhich is incorporated herein by reference). For example, in someembodiments, the Fc domain of a first interferon receptor Fc constructincludes S364H and F405A, and the Fc domain of a second interferonreceptor Fc construct includes Y349T and T394F.

The formation of heterodimeric soluble interferon receptors can also bepromoted by exploiting the structural similarity and sequence divergencebetween immunoglobulins from different classes. For example, the SEEDplatform uses the sequence divergence but structural similarity of theCH3 domains of IgG and IgA (Davis et al., (2010) Protein Eng. Des. Sel.23, 195-202, the entire contents of which is herein incorporated byreference). In some embodiments, the Fc domain of a first interferonreceptor Fc construct includes and IgG/A chimera and the Fc domain of asecond interferon receptor Fc construct includes and IgA/G chimera.

In other embodiments, the inability of IgG3 to bind protein A can beused for differential tagging of the Fc domain to enable efficientpurification of heterodimers (Davis et al., 2013, U.S. Pat. No.8,586,713, the entire contents of which are herein incorporated byreference). For example, in some embodiments, the Fc domain of ainterferon receptor Fc construct includes an H354R mutation.

In some embodiments, CH3 domain mutations can be introduced into IgG1,IgG2, and IgG4 constant regions to promote heterodimerization. In someembodiments, the Fc domain of a first interferon receptor Fc constructmay include a 407A substitution, and the Fc domain of a secondinterferon receptor Fc construct may include a 366V or 366M substitutionand a 409V substitution. In some embodiments, the Fc domain of a firstinterferon receptor Fc construct may include a 407A substitution as wellas one or more substitutions selected from the following group ofsubstitutions: 356G, 357D, 360D, 364Q, 364R, and 399M. In someembodiments, the Fc domain of a second interferon receptor Fc constructmay include a 366V or 366M substitution and a 409V substitution as wellas one or more substitutions selected from the following group ofsubstitutions: 345R, 347R, 349S, 366V, 370Y, and 399M. In someembodiments, the Fc domain of a second interferon receptor Fc constructmay include a 366V and a 409V substitution as well as one or moresubstitutions selected from the following group of substitutions: 345R,347R, 349S, 366V, 370Y, and 399M. In some embodiments, the Fc domain ofa second interferon receptor Fc construct may include a 366M and a 409Vsubstitution as well as one or more substitutions selected from thefollowing group of substitutions: 345R, 347R, 349S, 366V, 370Y, and399M. (see US2018/0009908 and Lewis et al., nature Biotechnology (2014)32(2), 191-198, both of which are incorporated by reference in theirentirety).

(ii) Appended IgG

In some embodiments, an Fc domain can be engineered to include twodifferent binding domains by appending either the amino and/or carboxyltermini of the Fc domain with one or more binding domains. In someembodiments, the one or more binding domains may be appended to the Fcdomain via a peptide linker.

In some embodiments, the binding domain is an IFNAR (e.g., IFNAR1 orIFNAR2), or variant or fragment thereof. In some embodiments, thebinding domain is the extracellular domain of IFNAR1 or IFNAR2.

(iii) Bispecific Fragments

In some embodiments, the soluble interferon receptor can lack some orall of the Fc domain. In some embodiments, binding domains and partialFc domains of a soluble interferon receptor that lacks some or all ofthe Fc domain may be connected via a peptide linker. In someembodiments, a soluble interferon receptor can be generated usingpolypeptide linkers to connect each of the domains of the solubleinterferon receptor (e.g., binding domains, Fc domains) and therebygenerate a single polypeptide chain.

In some embodiments, the binding domain is an IFNAR (e.g., IFNAR1 orIFNAR2), or variant or fragment thereof. In some embodiments, thebinding domain is the extracellular domain of IFNAR1 or IFNAR2.

(iv) Bispecific Fusion Proteins

In some embodiments, the binding domains of the soluble interferonreceptor are linked to other proteins to add additional functionality ofspecificity. In some embodiments, the binding domain is an IFNAR (e.g.,IFNAR1 or IFNAR2), or variant or fragment thereof. In some embodiments,the binding domain is the extracellular domain of IFNAR1 or IFNAR2.

(v) Bispecific Conjugates

In some embodiments, the binding domains of the soluble interferonreceptor are chemically conjugated to each other and/or to an Fc domain.In some embodiments, the binding domain is an IFNAR (e.g., IFNAR1 orIFNAR2), or variant or fragment thereof. In some embodiments, thebinding domain is the extracellular domain of IFNAR1 or IFNAR2.

(vi) IgG4 Fab Arm Exchange

In some embodiments, Fab arm exchange can be used to facilitate theheterodimerization of Fc domains. Fab arm exchange is a posttranslational modification of IgG4 antibodies that involves the thirdconstant domain of IgG4 in addition to the hinge region of IgG4 andrequires a reducing environment to be activated. (van der NeutKolfschoten et al. (2007) Science 317, 1554-1557, the entire contents ofwhich is incorporated herein by reference). IgG4 antibodies exchange Fabarms by swapping a heavy chain and attached light chain with a heavychain pair from another molecule, which results in bispecificantibodies. (van der Neut Kolfschoten et al. (2007)). In someembodiments, heterodimerization of the soluble interferon receptors ofthe disclosure can be promoted by replacing the CH3 domain in an IgG1 Fcwith the CH3 domain from an IgG4 Fc as well as replacing the IgG1 corehinge sequence with the IgG4 sequence (i.e., by replacing Pro228 withSer (P228S)).

(vii) Additional Alternative Scaffolds

In some embodiments, engineered proteins scaffolds (e.g., Affibody,DARPin, Adnectins) may be used to generate a molecule with at least twoIFNAR binding domains. In some embodiments, the binding domain is theextracellular domain of IFNAR1 or IFNAR2.

PK Moieties

In some embodiments, the soluble interferon receptor is operably coupledto a PK moiety, which serves as a scaffold as well as a means toincrease the serum half-life of the soluble interferon receptor.

Suitable PK moieties are well-known in the art and include, but are notlimited to, albumin, transferrin, Fc, and their variants, andpolyethylene glycol (PEG) and its derivatives. Suitable PK moietiesinclude, but are not limited to, HSA, or variants or fragments thereof,such as those disclosed in U.S. Pat. No. 5,876,969, WO 2011/124718, andWO 2011/0514789; Fc and Fc variants, such as those disclosed inWO2011/053982, WO 02/060955, WO 02/096948, WO05/047327, WO05/018572, andUS 2007/0111281; transferrin, or variants or fragments thereof, asdisclosed in U.S. Pat. Nos. 7,176,278 and 8,158,579; and PEG orderivatives, such as those disclosed in Zalipsky et al. (“Use ofFunctionalized Poly(Ethylene Glycols) for Modification of Polypeptides”in Polyethylene Glycol Chemistry: Biotechnical and BiomedicalApplications, J. M. Harris, Plenus Press, New York (1992)), and inZalipsky et al. Advanced Drug Reviews 1995:16: 157-182), and U.S. Pat.Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, 4,179,337,and 5,932,462 (the contents of the foregoing are incorporated herein byreference). It is within the abilities of the skilled artisan to useroutine methods to introduce PK moieties (e.g., cloning, conjugation)into the soluble interferon receptor of the invention.

In some embodiments, the PK moiety is HSA, which is naturallyaglycosylated.

In some embodiments, the PK moiety is a wild type Fc (SEQ ID NO: 26).

In certain embodiments, an Fc domain is altered or modified, e.g., byamino acid mutation (e.g., addition, deletion, or substitution). As usedherein, the term “Fc domain variant” refers to an Fc domain having atleast one amino acid modification, such as an amino acid substitution,as compared to the wild-type Fc from which the Fc domain is derived. Forexample, wherein the Fc domain is derived from a human IgG1 antibody, avariant comprises at least one amino acid mutation (e.g., substitution)as compared to a wild type amino acid at the corresponding position ofthe human IgG1 Fc region.

In some embodiments, the PK moiety is any of the Fc variants describedherein.

In some embodiments, the PK moiety is a wild type HST. In otherembodiments, the PK moiety is a HST with a mutations at N413 and/or N611and/or S12 (S12 is a potential O-linked glycosylation site), yielding aHST with altered glycosylation (i.e., HST N413S, HST N611S, HSTN413S/N611S and HST S12A/N413S/N611S).

Exemplary Soluble Interferon Receptors

The soluble interferon receptors of the disclosure can be configured toincorporate various interferon receptor Fc constructs. Likewise, theinterferon receptor Fc constructs of the invention can be configured toincorporate various domains.

For example, in one embodiment, the interferon receptor Fc construct mayinclude the IFNAR1 domain set forth in (SEQ ID NO: 11). In anotherembodiment, the interferon receptor Fc construct may include the IFNAR2domain set forth in (SEQ ID NO: 12). In another embodiment, theinterferon receptor Fc construct may include a linker domain. In anotherembodiment, the IFNAR1 domain is operatively coupled with a linkerdomain (e.g., a polypeptide linker) to a mutant Fc domain. In someembodiments, the IFNAR2 domain is operatively coupled with a linkerdomain (e.g., a polypeptide linker) to a mutant Fc domain. In someembodiments, the linker domain is a polypeptide linker (e.g., a Gly/Serlinker, e.g., (G₄S)_(n), wherein n is 1-10, 2-5, 1, 2, 3, 4, or 5). Insome embodiments, the polypeptide linker is about 1-50, about 5-40,about 10-30, or about 15-20 amino acids in length. In some aspects, thepolypeptide linker is about 20 amino acids or less, about 15 amino acidsor less, about 10 amino acids or less, or about 5 amino acids or less inlength. In some aspects, the polypeptide linker is 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29 or 30 amino acids in length. In another embodiment, theinterferon receptor Fc construct may include the (Gly₄Ser)₄ linkerdomain set forth in SEQ ID NO: 15. In another embodiment, the interferonreceptor Fc construct may include the (Gly₄Ser)₂ linker domain set forthin SEQ ID NO: 38. In another embodiment, the interferon receptor Fcconstruct does not include a linker domain.

In another embodiment, the interferon receptor Fc construct may includea leader sequence set forth in SEQ ID NO: 13.

In some embodiments, the interferon receptor Fc construct may include anIgG1 Fc domain comprising mutations C220S, P238S, P331S, and T350V,T366L, K392L, and T394W as set forth in (SEQ ID NO: 10). In someembodiments, the interferon receptor Fc construct may include an IgG1 Fcdomain comprising mutations C220S, P238S, P331S, and T350V, L351Y,F405A, and Y407V as set forth in (SEQ ID NO: 9). In some embodiments,the interferon Fc receptor construct may include an IgG1 Fc domaincomprising the mutation T366Y as set forth in SEQ ID NO: 106, andoptionally, one or more mutations selected from C220S, P238S, and P331S.In some embodiments, the interferon Fc receptor construct may include anIgG1 Fc domain comprising the mutation Y407T as set forth in SEQ ID NO:107, and optionally, one or more mutations selected from C220S, P238S,and P331S. In some embodiments, the interferon Fc receptor construct mayinclude an IgG1 Fc domain comprising the mutation T366W as set forth inSEQ ID NO: 108, and optionally, one or more mutations selected fromC220S, P238S, and P331S. In some embodiments, the interferon Fcconstruct may include an IgG1 Fc domain comprising mutations T366S,L368A, and Y407V as set forth in SEQ ID NO: 109, and optionally, one ormore mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon Fc receptor construct comprises amutant IgG4 Fc domain comprising mutation S228P, F296Y, E356K, R409K,H435R and L445P according to EU numbering. In some embodiments, theheterodimer comprises a mutant IgG4 Fc domain comprising mutationsT366S, L368A and Y407V, according to EU numbering. In some embodiments,the interferon Fc construct comprises the IgG4 Fc domain comprisingmutations S228P, F234A, L235A, L445P (EU) and K478del (Kabat) as setforth in SEQ ID NO: 113. In some embodiments, the interferon Fcconstruct comprises the IgG4 Fc domain with mutations F296Y, E356K,R409K, and H435R (EU) as set forth in SEQ ID NO: 116. In someembodiments, the interferon Fc construct comprises the IgG4 Fc domainwith mutations F296Y, R409K, and K439E (EU) as set forth in SEQ ID NO:117. In some embodiments, the interferon Fc construct comprises the IgG4Fc domain with mutations S228P, F296Y, E356K, R409K, H435R, L445P,G446del (EU) and K478del(Kabat) as set forth in SEQ ID NO: 118. In someembodiments, the interferon Fc construct comprises the IgG4 Fc domainwith mutations S228P, F296Y, R409K, K439E, L445P, G446del (EU) andK478del(Kabat) as set forth in SEQ ID NO: 119.

It will be understood to the skilled artisan that these individualdomains can be operably coupled to each other in any order to form aninterferon receptor Fc construct that is enzymatically active. Forexample, as detailed in the specific examples below, an IFNAR1extracellular domain can be operatively coupled to an Fc domain via a(Gly₄Ser)₄ linker domain. In another example, an IFNAR2 extracellulardomain can be operatively coupled to an Fc domain via a (Gly₄Ser)₄linker domain. In another example, an IFNAR1 extracellular domain can beoperatively coupled to an Fc domain via a (Gly₄Ser)₂ linker domain. Inanother example, an IFNAR2 extracellular domain can be operativelycoupled to an Fc domain via a (Gly₄Ser)₂ linker domain. In anotherexample, an IFNAR1 extracellular domain can be operably coupled to an Fcdomain. In another example, an IFNAR2 extracellular domain can beoperably coupled to an Fc domain. Various other configurations arepossible, with non-limiting exemplary configurations disclosed herein,in FIG. 1 and in the Sequence Table.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations C220S, P238S, P331S, and T350V, T366L, K392L, andT394W via a linker domain, such as a (Gly₄Ser)₄ linker domain, with orwithout a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations C220S, P238S, P331S, and T350V, T366L, K392L, andT394W via a linker domain, such as a (Gly₄Ser)₂ linker domain, with orwithout a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations C220S, P238S, P331S, and T350V, T366L, K392L, andT394W without a linker domain, with or without a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations C220S, P238S, P331S, and T350V, T366L, K392L, andT394W via a linker domain, such as a (Gly₄Ser)₄ linker domain, with orwithout a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations C220S, P238S, P331S, and T350V, T366L, K392L, andT394W via a linker domain, such as a (Gly₄Ser)₂ linker domain, with orwithout a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations C220S, P238S, P331S, and T350V, T366L, K392L, andT394W without a linker domain, with or without a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations C220S, P238S, P331S, and T350V, L351Y, F405A, andY407V via a linker domain, such as a (Gly₄Ser)₄ linker domain, with orwithout a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations C220S, P238S, P331S, and T350V, L351Y, F405A, andY407V via a linker domain, such as a (Gly₄Ser)₂ linker domain, with orwithout a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations C220S, P238S, P331S, and T350V, L351Y, F405A, andY407V without a linker domain, with or without a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations C220S, P238S, P331S, and T350V, L351Y, F405A, andY407V via a linker domain, such as a (Gly₄Ser)₄ linker domain, with orwithout a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations C220S, P238S, P331S, and T350V, L351Y, F405A, andY407V via a linker domain, such as a (Gly₄Ser)₂ linker domain, with orwithout a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations C220S, P238S, P331S, and T350V, L351Y, F405A, andY407V without a linker domain, with or without a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366Y via a linker domain, such as a (Gly₄Ser)₄linker domain, with or without a leader sequence, and optionally, one ormore mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366Y via a linker domain, such as a (Gly₄Ser)₂linker domain, with or without a leader sequence, and optionally, one ormore mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366Y without a linker domain, with or without aleader sequence, and optionally, one or more mutations selected fromC220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366Y via a linker domain, such as a (Gly₄Ser)₄linker domain, with or without a leader sequence, and optionally, one ormore mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366Y via a linker domain, such as a (Gly₄Ser)₂linker domain, with or without a leader sequence, and optionally, one ormore mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366Y without a linker domain, with or without aleader sequence, and optionally, one or more mutations selected fromC220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation Y407T via a linker domain, such as a (Gly₄Ser)₄linker domain, with or without a leader sequence, and optionally, one ormore mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation Y407T via a linker domain, such as a (Gly₄Ser)₂linker domain, with or without a leader sequence, and optionally, one ormore mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation Y407T without a linker domain, with or without aleader sequence, and optionally, one or more mutations selected fromC220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation Y407T via a linker domain, such as a (Gly₄Ser)₄linker domain, with or without a leader sequence, and optionally, one ormore mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation Y407T via a linker domain, such as a (Gly₄Ser)₂linker domain, with or without a leader sequence, and optionally, one ormore mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation Y407T without a linker domain, with or without aleader sequence, and optionally, one or more mutations selected fromC220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366W via a linker domain, such as a (Gly₄Ser)₄linker domain, with or without a leader sequence, and optionally, one ormore mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366W via a linker domain, such as a (Gly₄Ser)₂linker domain, with or without a leader sequence, and optionally, one ormore mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366W without a linker domain, with or without aleader sequence, and optionally, one or more mutations selected fromC220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366W via a linker domain, such as a (Gly₄Ser)₄linker domain, with or without a leader sequence, and optionally, one ormore mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366W via a linker domain, such as a (Gly₄Ser)₂linker domain, with or without a leader sequence, and optionally, one ormore mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366W without a linker domain, with or without aleader sequence, and optionally, one or more mutations selected fromC220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations T366S, L368A, and Y407V via a linker domain, suchas a (Gly₄Ser)₄ linker domain, with or without a leader sequence, andoptionally, one or more mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations T366S, L368A, and Y407V via a linker domain, suchas a (Gly₄Ser)₂ linker domain, with or without a leader sequence, andoptionally, one or more mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations T366S, L368A, and Y407V without a linker domain,with or without a leader sequence, and optionally, one or more mutationsselected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations T366S, L368A, and Y407V via a linker domain, suchas a (Gly₄Ser)₄ linker domain, with or without a leader sequence, andoptionally, one or more mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations T366S, L368A, and Y407V via a linker domain, suchas a (Gly₄Ser)₂ linker domain, with or without a leader sequence, andoptionally, one or more mutations selected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations T366S, L368A, and Y407V without a linker domain,with or without a leader sequence, and optionally, one or more mutationsselected from C220S, P238S, and P331S.

In some embodiments, the interferon receptor Fc construct comprises aIFNAR1 operably coupled to an IgG4 Fc domain comprising mutations F296Y,E356K, R409K, and H435R via a linker domain, such as a (Gly₄Ser)₄ linkerdomain, with or without a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises aIFNAR1 operably coupled to an IgG4 Fc domain comprising mutations F296Y,E356K, R409K, and H435R via a linker domain, such as a (Gly₄Ser)₂ linkerdomain, with or without a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises aIFNAR1 operably coupled to an IgG4 Fc domain comprising mutations F296Y,E356K, R409K, and H435R without a linker domain, with or without aleader sequence.

In some embodiments, the interferon receptor Fc construct comprises aIFNAR2 operably coupled to an IgG4 Fc domain comprising mutations F296Y,E356K, R409K, and H435R via a linker domain, such as a (Gly₄Ser)₄ linkerdomain, with or without a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises aIFNAR2 operably coupled to an IgG4 Fc domain comprising mutations F296Y,E356K, R409K, and H435R via a linker domain, such as a (Gly₄Ser)₂ linkerdomain, with or without a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises aIFNAR2 operably coupled to an IgG4 Fc domain comprising mutations F296Y,E356K, R409K, and H435R without a linker domain, with or without aleader sequence.

In some embodiments, the interferon receptor Fc construct comprises aIFNAR1 operably coupled to an IgG4 Fc domain comprising mutations F296Y,R409K, and K439E via a linker domain, such as a (Gly₄Ser)₄ linkerdomain, with or without a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises aIFNAR1 operably coupled to an IgG4 Fc domain comprising mutations F296Y,R409K, and K439E via a linker domain, such as a (Gly₄Ser)₂ linkerdomain, with or without a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises aIFNAR1 operably coupled to an IgG4 Fc domain comprising mutations F296Y,R409K, and K439E without a linker domain, with or without a leadersequence.

In some embodiments, the interferon receptor Fc construct comprises aIFNAR2 operably coupled to an IgG4 Fc domain comprising mutations F296Y,R409K, and K439E via a linker domain, such as a (Gly₄Ser)₄ linkerdomain, with or without a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises aIFNAR2 operably coupled to an IgG4 Fc domain comprising mutations F296Y,R409K, and K439E via a linker domain, such as a (Gly₄Ser)₂ linkerdomain, with or without a leader sequence.

In some embodiments, the interferon receptor Fc construct comprises aIFNAR2 operably coupled to an IgG4 Fc domain comprising mutations F296Y,R409K, and K439E without a linker domain, with or without a leadersequence.

In some embodiments, the interferon receptor Fc construct comprises theamino acid sequence set forth as SEQ ID NO: 1, with or without a leadersequence, and nucleic acids encoding the amino acid sequence set forthas SEQ ID NO:1.

In some embodiments, the interferon receptor Fc construct comprises theamino acid sequence set forth as SEQ ID NO: 2, with or without a leadersequence, and nucleic acids encoding the amino acid sequence set forthas SEQ ID NO:2.

In some embodiments, the interferon receptor Fc construct comprises theamino acid sequence set forth as SEQ ID NO: 3, with or without a leadersequence, and nucleic acids encoding the amino acid sequence set forthas SEQ ID NO:3.

In some embodiments, the interferon receptor Fc construct comprises theamino acid sequence set forth as SEQ ID NO: 4, with or without a leadersequence, and nucleic acids encoding the amino acid sequence set forthas SEQ ID NO:4.

In some embodiments, the interferon receptor Fc construct comprises theamino acid sequence set forth as SEQ ID NO: 52, with or without a leadersequence, and nucleic acids encoding the amino acid sequence set forthas SEQ ID NO: 52.

In some embodiments, the interferon receptor Fc construct comprises theamino acid sequence set forth as SEQ ID NO: 54, with or without a leadersequence, and nucleic acids encoding the amino acid sequence set forthas SEQ ID NO: 54.

In some embodiments, the interferon receptor Fc construct comprises theamino acid sequence set forth as SEQ ID NO: 56, with or without a leadersequence, and nucleic acids encoding the amino acid sequence set forthas SEQ ID NO: 56.

In some embodiments, the interferon receptor Fc construct comprises theamino acid sequence set forth as SEQ ID NO: 58, with or without a leadersequence, and nucleic acids encoding the amino acid sequence set forthas SEQ ID NO: 58.

In some embodiments, the interferon receptor Fc constructs dimerize toform a soluble interferon receptor, such as a heterodimeric solubleinterferon receptor. For example, in some embodiments, the solubleinterferon receptor comprises a heterodimer comprising an interferonreceptor Fc construct comprising an IFNAR1 extracellular domain operablycoupled to an IgG1 Fc domain comprising mutations C220S, P238S, P331S,and T350V, T366L, K392L, and T394W via a linker domain, such as a(Gly₄Ser)₄ linker domain, with or without a leader sequence, and aninterferon receptor Fc construct comprising an IFNAR2 extracellulardomain operably coupled to an IgG1 Fc domain comprising mutations C220S,P238S, P331S, and T350V, L351Y, F405A, and Y407V via a linker domain,such as a (Gly₄Ser)₄ linker domain, with or without a leader sequence.For example, in some embodiments, the soluble interferon receptorcomprises a heterodimer comprising an interferon receptor Fc constructcomprising an IFNAR2 extracellular domain operably coupled to an IgG1 Fcdomain comprising mutations C220S, P238S, P331S, and T350V, T366L,K392L, and T394W via a linker domain, such as a (Gly₄Ser)₄ linkerdomain, with or without a leader sequence, and an interferon receptor Fcconstruct comprising an IFNAR1 extracellular domain operably coupled toan IgG1 Fc domain comprising mutations C220S, P238S, P331S, and T350V,L351Y, F405A, and Y407V via a linker domain, such as a (Gly₄Ser)₄ linkerdomain, with or without a leader sequence.

In some embodiments, the soluble interferon receptor comprises aheterodimer comprising an interferon receptor Fc construct comprising anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations C220S, P238S, P331S, and T350V, T366L, K392L, andT394W via a linker domain, such as a (Gly₄Ser)₂ linker domain, with orwithout a leader sequence, and an interferon receptor Fc constructcomprising an IFNAR2 extracellular domain operably coupled to an IgG1 Fcdomain comprising mutations C220S, P238S, P331S, and T350V, L351Y,F405A, and Y407V via a linker domain, such as a (Gly₄Ser)₂ linkerdomain, with or without a leader sequence. For example, in someembodiments, the soluble interferon receptor comprises a heterodimercomprising an interferon receptor Fc construct comprising an IFNAR2extracellular domain operably coupled to an IgG1 Fc domain comprisingmutations C220S, P238S, P331S, and T350V, T366L, K392L, and T394W via alinker domain, such as a (Gly₄Ser)₂ linker domain, with or without aleader sequence, and an interferon receptor Fc construct comprising anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations C220S, P238S, P331S, and T350V, L351Y, F405A, andY407V via a linker domain, such as a (Gly₄Ser)₂ linker domain, with orwithout a leader sequence.

In some embodiments, the soluble interferon receptor comprises aheterodimer comprising an interferon receptor Fc construct comprising anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations C220S, P238S, P331S, and T350V, T366L, K392L, andT394W without a linker domain, with or without a leader sequence, and aninterferon receptor Fc construct comprising an IFNAR2 extracellulardomain operably coupled to an IgG1 Fc domain comprising mutations C220S,P238S, P331S, and T350V, L351Y, F405A, and Y407V without a linkerdomain, with or without a leader sequence. For example, in someembodiments, the soluble interferon receptor comprises a heterodimercomprising an interferon receptor Fc construct comprising an IFNAR2extracellular domain operably coupled to an IgG1 Fc domain comprisingmutations C220S, P238S, P331S, and T350V, T366L, K392L, and T394Wwithout a linker domain, with or without a leader sequence, and aninterferon receptor Fc construct comprising an IFNAR1 extracellulardomain operably coupled to an IgG1 Fc domain comprising mutations C220S,P238S, P331S, and T350V, L351Y, F405A, and Y407V without a linkerdomain, with or without a leader sequence.

In some embodiments, the soluble interferon receptor comprises aheterodimer comprising an interferon receptor Fc construct comprising anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366Y via a linker domain, such as a (Gly₄Ser)₄linker domain, with or without a leader sequence, and an interferonreceptor Fc construct comprising an IFNAR2 extracellular domain operablycoupled to an IgG1 Fc domain comprising mutation Y407T via a linkerdomain, such as a (Gly₄Ser)₄ linker domain, with or without a leadersequence. For example, in some embodiments, the soluble interferonreceptor comprises a heterodimer comprising an interferon receptor Fcconstruct comprising an IFNAR1 extracellular domain operably coupled toan IgG1 Fc domain comprising mutation Y407T via a linker domain, such asa (Gly₄Ser)₄ linker domain, with or without a leader sequence, and aninterferon receptor Fc construct comprising an IFNAR2 extracellulardomain operably coupled to an IgG1 Fc domain comprising mutation T366Yvia a linker domain, such as a (Gly₄Ser)₄ linker domain, with or withouta leader sequence.

In some embodiments, the soluble interferon receptor comprises aheterodimer comprising an interferon receptor Fc construct comprising anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366Y via a linker domain, such as a (Gly₄Ser)₂linker domain, with or without a leader sequence, and an interferonreceptor Fc construct comprising an IFNAR2 extracellular domain operablycoupled to an IgG1 Fc domain comprising mutation Y407T via a linkerdomain, such as a (Gly₄Ser)₂ linker domain, with or without a leadersequence. For example, some embodiments, the soluble interferon receptorFc construct comprises a heterodimer comprising an interferon receptorFc construct comprising an IFNAR1 extracellular domain operably coupledto an IgG1 Fc domain comprising mutation Y407T via a linker domain, suchas a (Gly₄Ser)₂ linker domain, with or without a leader sequence, and aninterferon receptor Fc construct comprising an IFNAR2 extracellulardomain operably coupled to an IgG1 Fc domain comprising mutation T366Yvia a linker domain, such as a (Gly₄Ser)₂ linker domain, with or withouta leader sequence.

In some embodiments, the soluble interferon receptor comprises aheterodimer comprising an interferon receptor Fc construct comprising anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366Y without a linker domain, with or without aleader sequence, and an interferon receptor Fc construct comprising anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation Y407T without a linker domain, with or without aleader sequence. For example, in some embodiments, the solubleinterferon receptor Fc construct comprises a heterodimer comprising aninterferon receptor Fc construct comprising an IFNAR1 extracellulardomain operably coupled to a Fc domain comprising mutation Y407T withouta linker domain, with or without a leader sequence, and an interferonreceptor Fc construct comprises an IFNAR2 extracellular domain operablycoupled to an IgG1 Fc domain comprising mutation T366Y without a linkerdomain, with or without a leader sequence.

In some embodiments, the soluble interferon receptor comprises aheterodimer comprising an interferon receptor Fc construct comprising anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366W via a linker domain, such as a (Gly₄Ser)₄linker domain, with or without a leader sequence, and an interferonreceptor Fc construct comprising an IFNAR2 extracellular domain operablycoupled to an IgG1 Fc domain comprising mutations T366S, L368A, andY407V via a linker domain, such as a (Gly₄Ser)₄ linker domain, with orwithout a leader sequence. For example, in some embodiments, the solubleinterferon receptor comprises a heterodimer comprising an interferonreceptor Fc construct comprising an IFNAR1 extracellular domain operablycoupled to an IgG1 Fc domain comprising mutations T366S, L368A, andY407V via a linker domain, such as a (Gly₄Ser)₄ linker domain, with orwithout a leader sequence, and an interferon receptor Fc constructcomprising an IFNAR2 extracellular domain operably coupled to an IgG1 Fcdomain comprising mutation T366W via a linker domain, such as a(Gly₄Ser)₄ linker domain, with or without a leader sequence.

In some embodiments, the soluble interferon receptor comprises aheterodimer comprising an interferon receptor Fc construct comprising anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366W via a linker domain, such as a (Gly₄Ser)₂linker domain, with or without a leader sequence, and an interferonreceptor Fc construct comprising an IFNAR2 extracellular domain operablycoupled to an IgG1 Fc domain comprising mutations T366S, L368A, andY407V via a linker domain, such as a (Gly₄Ser)₂ linker domain, with orwithout a leader sequence. For example, in some embodiments, the solubleinterferon receptor comprises a heterodimer comprising an interferonreceptor Fc construct comprising an IFNAR1 extracellular domain operablycoupled to an IgG1 Fc domain comprising mutations T366S, L368A, andY407V via a linker domain, such as a (Gly₄Ser)₂ linker domain, with orwithout a leader sequence, and an interferon receptor Fc constructcomprising an IFNAR2 extracellular domain operably coupled to an IgG1 Fcdomain comprising mutation T366W via a linker domain, such as a(Gly₄Ser)₂ linker domain, with or without a leader sequence.

In some embodiments, the soluble interferon receptor comprises aheterodimer comprising an interferon receptor Fc construct comprising anIFNAR1 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutation T366W without a linker domain, with or without aleader sequence, and an interferon receptor Fc construct comprising anIFNAR2 extracellular domain operably coupled to an IgG1 Fc domaincomprising mutations T366S, L368A, and Y407V without a linker domain,with or without a leader sequence. For example, in some embodiments, thesoluble interferon receptor comprises a heterodimer comprising aninterferon receptor Fc construct comprising an IFNAR1 extracellulardomain operably coupled to an IgG1 Fc domain comprising mutations T366S,L368A, and Y407V without a linker domain, with or without a leadersequence, and an interferon receptor Fc construct comprising an IFNAR2extracellular domain operably coupled to an IgG1 Fc domain comprisingmutation T366W without a linker domain, with or without a leadersequence.

In some embodiments, the soluble interferon receptor comprises aheterodimer comprising an interferon receptor Fc construct comprising aIFNAR1 operably coupled to an IgG4 Fc domain comprising mutations F296Y,E356K, R409K, and H435R via a linker domain, such as a (Gly₄Ser)₄ linkerdomain, with or without a leader sequence, and an interferon receptor Fcconstruct comprising a IFNAR2 operably coupled to an IgG4 Fc domaincomprising mutations F296Y, R409K, and K439E via a linker domain, suchas a (Gly₄Ser)₄ linker domain, with or without a leader sequence. Forexample, in some embodiments, the soluble interferon receptor comprisesa heterodimer comprising an interferon receptor Fc construct comprisinga IFNAR1 operably coupled to an IgG4 Fc domain comprising mutationsF296Y, R409K, and K439E via a linker domain, such as a (Gly₄Ser)₄ linkerdomain, with or without a leader sequence, and an interferon receptor Fcconstruct comprising a IFNAR2 operably coupled to an IgG4 Fc domaincomprising mutations F296Y, E356K, R409K, and H435R via a linker domain,such as a (Gly₄Ser)₄ linker domain, with or without a leader sequence.

In some embodiments, the soluble interferon receptor comprises aheterodimer comprising an interferon receptor Fc construct comprising aIFNAR1 operably coupled to an IgG4 Fc domain comprising mutations F296Y,E356K, R409K, and H435R via a linker domain, such as a (Gly₄Ser)₂ linkerdomain, with or without a leader sequence, and an interferon receptor Fcconstruct comprising a IFNAR2 operably coupled to an IgG4 Fc domaincomprising mutations F296Y, R409K, and K439E via a linker domain, suchas a (Gly₄Ser)₂ linker domain, with or without a leader sequence. Forexample, in some embodiments, the soluble interferon receptor Fcconstruct comprises a heterodimer comprising an interferon receptor Fcconstruct comprising a IFNAR1 operably coupled to an IgG4 Fc domaincomprising mutations F296Y, R409K, and K439E via a linker domain, suchas a (Gly₄Ser)₂ linker domain, with or without a leader sequence, and aninterferon receptor Fc construct comprising a IFNAR2 operably coupled toan IgG4 Fc domain comprising mutations F296Y, E356K, R409K, and H435Rvia a linker domain, such as a (Gly₄Ser)₂ linker domain, with or withouta leader sequence.

In some embodiments, the soluble interferon receptor comprises aheterodimer comprising an interferon receptor Fc construct comprising aIFNAR1 operably coupled to an IgG4 Fc domain comprising mutations F296Y,E356K, R409K, and H435R without a linker domain, with or without aleader sequence, and an interferon receptor Fc construct comprising aIFNAR2 operably coupled to an IgG4 Fc domain comprising mutations F296Y,R409K, and K439E without a linker domain, with or without a leadersequence. For example, in some embodiments, the soluble interferonreceptor Fc construct comprises a heterodimer comprising an interferonreceptor Fc construct comprising a IFNAR1 operably coupled to an IgG4 Fcdomain comprising mutations F296Y, R409K, and K439E without a linkerdomain, with or without a leader sequence, and an interferon receptor Fcconstruct comprising a IFNAR2 operably coupled to an IgG4 Fc domaincomprising mutations F296Y, E356K, R409K, and H435R without a linkerdomain, with or without a leader sequence.

In some embodiments, a soluble interferon receptor is a heterodimercomprising a polypeptide comprising the amino acid sequence set forth inSEQ ID NO: 1 and a polypeptide comprising the amino acid sequence setfor in SEQ ID NO: 4.

In some embodiments, a soluble interferon receptor is a heterodimercomprising a polypeptide comprising the amino acid sequence set forth inSEQ ID NO: 2 and a polypeptide comprising the amino acid sequence setfor in SEQ ID NO: 3.

In some embodiments, a soluble interferon receptor is a heterodimercomprising a polypeptide comprising the amino acid sequence set forth inSEQ ID NO: 52 and a polypeptide comprising the amino acid sequence setfor in SEQ ID NO: 54.

In some embodiments, a soluble interferon receptor is a heterodimercomprising a polypeptide comprising the amino acid sequence set forth inSEQ ID NO: 56 and a polypeptide comprising the amino acid sequence setfor in SEQ ID NO: 58.

In some embodiments, a soluble interferon receptor is a heterodimercomprising a polypeptide comprising the amino acid sequence set forth inSEQ ID NO: 40 and a polypeptide comprising the amino acid sequence setfor in SEQ ID NO: 43.

In some embodiments, a soluble interferon receptor is a heterodimercomprising a polypeptide comprising the amino acid sequence set forth inSEQ ID NO: 41 and a polypeptide comprising the amino acid sequence setfor in SEQ ID NO: 42.

In some embodiments, a soluble interferon receptor is a heterodimercomprising a polypeptide comprising the amino acid sequence set forth inSEQ ID NO: 53 and a polypeptide comprising the amino acid sequence setfor in SEQ ID NO: 55.

In some embodiments, a soluble interferon receptor is a heterodimercomprising a polypeptide comprising the amino acid sequence set forth inSEQ ID NO: 57 and a polypeptide comprising the amino acid sequence setfor in SEQ ID NO: 59.

In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 1.In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 2.In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 3.In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 4.In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 52.In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 54.In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 56.In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 58.In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 40.In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 43.In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 41.In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 42.In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 53.In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 55.In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 57.In some embodiments, an interferon receptor Fc construct comprises apolypeptide having an amino acid sequence at least 70% identical, suchas 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 59.In some embodiments, the polypeptide comprises an amino acid sequenceselected from any one of SEQ ID NOs: 1-4, 52, 54, 56, 58, 40, 43, 41,42, 43, 55, 57 or 59.

In some embodiments, the foregoing interferon receptor Fc constructshave a leader sequence. In some embodiments, the foregoing interferonreceptor Fc constructs do not have a leader sequence.

It will be understood by one of ordinary skill that the leader andlinker sequences are optional and are not limited to those described inthe embodiments above. For example, the IFNAR domains (e.g., IFNAR1,IFNAR2) can be directly fused to the N- and/or C-terminus of an Fcdomain, or variant or fragment thereof; the leader domain can be any ofthose known in the art to be useful for its intended purpose, e.g., toincrease protein expression and/or secretion (e.g., MDWTWRILFLVAAATGTHA;SEQ ID NO: 13); the linker can be any linker known in the art, e.g.,(Gly4Ser)n, NLG (VDGASSPVNVSSPSVQDI; SEQ ID NO: 18), LE,thrombin-sensitive disulphide cyclopeptide linker,LEA(EAAAK)₄ALEA(EAAAK)₄ (SEQ ID NO: 19), or an in vivo cleavabledisulphide linker, as described herein. It will also be understood thatit is within the abilities of a skilled artisan to make thecorresponding changes to the amino acid sequences of the interferonreceptor Fc constructs using routine cloning and recombination methods.

Methods of Making Soluble Interferon Receptors

The interferon receptor Fc constructs of this disclosure largely may bemade in transformed or transfected host cells using recombinant DNAtechniques. To do so, a recombinant DNA molecule coding for the peptideis prepared. Methods of preparing such DNA molecules are well known inthe art. For instance, sequences coding for the interferon receptor Fcconstructs could be excised from DNA using suitable restriction enzymes.Alternatively, the DNA molecule could be synthesized using chemicalsynthesis techniques, such as the phosphoramidate method. Also, acombination of these techniques could be used.

The invention also includes a vector capable of expressing theinterferon receptor Fc constructs in an appropriate host. The vectorcomprises the DNA molecule that codes for the interferon receptor Fcconstruct operably coupled to appropriate expression control sequences.Methods of affecting this operative linking, either before or after theDNA molecule is inserted into the vector, are well known. Expressioncontrol sequences include promoters, activators, enhancers, operators,ribosomal nuclease domains, start signals, stop signals, cap signals,polyadenylation signals, and other signals involved with the control oftranscription or translation.

The resulting vector having the DNA molecule thereon is used totransform or transfect an appropriate host. In some embodiments, thesoluble interferon receptors of the disclosure may be made byco-transfecting or co-transforming two or more expression vectorscomprising DNA that codes for an interferon receptor Fc construct intoan appropriate host. This transformation or transfection may beperformed using methods well known in the art.

Any of a large number of available and well-known host cells may be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the interferon receptor Fc constructs encoded by the DNA molecule,rate of transformation or transfection, ease of recovery of theinterferon receptor Fc constructs, expression characteristics,bio-safety and costs. A balance of these factors must be struck with theunderstanding that not all hosts may be equally effective for theexpression of a particular DNA sequence. Within these generalguidelines, useful microbial hosts include bacteria (such as E. coli),yeast (such as Saccharomyces) and other fungi, insects, plants,mammalian (including human) cells in culture, or other hosts known inthe art. In some embodiments, the interferon receptor Fc constructs areproduced in CHO cells.

Next, the transformed or transfected host is cultured and purified. Hostcells may be cultured under conventional fermentation or cultureconditions so that the desired compounds are expressed. Suchfermentation and culture conditions are well known in the art. Finally,the interferon receptor Fc constructs are purified from culture bymethods well known in the art.

The compounds may also be made by synthetic methods. For example, solidphase synthesis techniques may be used. Suitable techniques are wellknown in the art, and include those described in Merrifield (1973),Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.);Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al., BiochemIntl 1985; 10: 394-414; Stewart and Young (1969), Solid Phase PeptideSynthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins(3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.)2: 257-527. In some embodiments, compounds that contain derivatizedpeptides or which contain non-peptide groups may be synthesized bywell-known organic chemistry techniques.

Other methods are of molecule expression/synthesis are generally knownin the art to one of ordinary skill.

Soluble Interferon Receptors with Altered Glycosylation

Glycosylation (e.g., 0-lined or N-linked glycosylation) can impact theserum half-life of the soluble interferon receptor of the disclosure by,e g, minimizing their removal from circulation by mannose andasialoglycoprotein receptors and other lectin-like receptors.Accordingly, in some embodiments, the soluble interferon receptors ofthe disclosure are prepared in aglycosylated, deglycosylated, orunderglycosylated form. Preferably, N-linked glycosylation is alteredand the soluble interferon receptor is aglycosyated.

In some embodiments, all asparagine residues in a soluble interferonreceptor that conform to the Asn-X-Ser/Thr (X can be any other naturallyoccurring amino acid except Pro) consensus are mutated to residues thatdo not serve as acceptors of N-linked glycosylation (e.g., serine,glutamine), thereby eliminating glycosylation of the soluble interferonreceptor when synthesized in a cell that glycosylates proteins.

In some embodiments, soluble interferon receptor lacking N-linkedglycosylation sites are produced in mammalian cells. In one embodiment,the mammalian cell is a CHO cell. Accordingly, in a specific embodiment,an aglycosylated soluble interferon receptor is produced in a CHO cell.

In other embodiments, a reduction or lack of N-glycosylation is achievedby, e.g., producing soluble interferon receptors in a host (e.g.,bacteria such as E. coli), mammalian cells engineered to lack one ormore enzymes important for glycosylation, or mammalian cells treatedwith agents that prevent glycosylation, such as tunicamycin (aninhibitor of Dol-PP-GlcNAc formation).

In some embodiments, the soluble interferon receptors are produced inlower eukaryotes engineered to produce glycoproteins with complexN-glycans, rather than high mannose type sugars (see, e.g.,US2007/0105127).

In some embodiments, glycosylated soluble interferon receptors (e.g.,those produced in mammalian cells such as CHO cells) are treatedchemically or enzymatically to remove one or more carbohydrate residues(e.g., one or more mannose, fucose, and/or N-acetylglucosamine residues)or to modify or mask one or more carbohydrate residues. Suchmodifications or masking may reduce binding of the soluble interferonreceptors to mannose receptors, and/or asialoglycoprotein receptors,and/or other lectin-like receptors. Chemical deglycosylation can beachieved by treating a soluble interferon receptor with trifluoromethanesulfonic acid (TFMS), as disclosed in, e.g., Sojar et al., JBC 1989;264:2552-9 and Sojar et al., Methods Enzymol 1987; 138:341-50, or bytreating with hydrogen fluoride, as disclosed in Sojar et al. (1987,supra). Enzymatic removal of N-linked carbohydrates from solubleinterferon receptor can be achieved by treating a soluble interferonreceptor with protein N-glycosidase (PNGase) A or F, as disclosed inThotakura et al. (Methods Enzymol 1987; 138:350-9). Other art-recognizedcommercially available deglycosylating enzymes that are suitable for useinclude endo-alpha-N-acetyl-galactosaminidase, endoglycosidase F1,endoglycosidase F2, endoglycosidase F3, and endoglycosidase H. In someembodiments, one or more of these enzymes can be used to deglycosylatethe soluble interferon receptors of the disclosure. Alternative methodsfor deglycosylation are disclosed in, e.g., U.S. Pat. No. 8,198,063.

In some embodiments, the soluble interferon receptor are partiallydeglycosylated. Partial deglycosylation can be achieved by treating thesoluble interferon receptor with an endoglycosidase (e.g.,endoglycosidase H), which cleaves N-linked high mannose carbohydrate butnot complex type carbohydrates, leaving a single GlcNAc residue linkedto the asparagine. Soluble interferon receptor treated withendoglycosidase H will lack high mannose carbohydrates, resulting in areduced interaction with the hepatic mannose receptor. Although thisreceptor recognizes terminal GlcNAc, the probability of a productiveinteraction with the single GlcNAc on the protein surface is not asgreat as with an intact high mannose structure.

In other embodiments, glycosylation of a soluble interferon receptor ismodified, e.g., by oxidation, reduction, dehydration, substitution,esterification, alkylation, sialylation, carbon-carbon bond cleavage, orthe like, to reduce clearance of the soluble interferon receptors fromblood. In some embodiments, the soluble interferon receptors are treatedwith periodate and sodium borohydride to modify the carbohydratestructure. Periodate treatment oxidizes vicinal diols, cleaving thecarbon-carbon bond and replacing the hydroxyl groups with aldehydegroups; borohydride reduces the aldehydes to hydroxyls. Many sugarresidues include vicinal diols and, therefore, are cleaved by thistreatment. Prolonged serum half-life with periodate and sodiumborohydride is exemplified by the sequential treatment of the lysosomalenzyme β-glucuronidase with these agents (see, e.g., Houba et al. (1996)Bioconjug Chem 1996:7:606-11; Stahl et al. PNAS 1976; 73:4045-9; Achordet al. Pediat. Res 1977; 11:816-22; Achord et al. Cell 1978; 15:269-78).A method for treatment with periodate and sodium borohydride isdisclosed in Hickman et al., BBRC 1974; 57:55-61. A method for treatmentwith periodate and cyanoborohydride, which increases the serum half-lifeand tissue distribution of ricin, is disclosed in Thorpe et al. Eur JBiochem 1985; 147:197-206.

In one embodiment, the carbohydrate structures of a soluble interferonreceptor can be masked by addition of one or more additional moieties(e.g., carbohydrate groups, phosphate groups, alkyl groups, etc.) thatinterfere with recognition of the structure by a mannose orasialoglycoprotein receptor or other lectin-like receptors.

In some embodiments, one or more potential glycosylation sites areremoved by mutation of the nucleic acid encoding the soluble interferonreceptor, thereby reducing glycosylation (underglycosylation) of thesoluble interferon receptor when synthesized in a cell that glycosylatesproteins, e.g., a mammalian cell such as a CHO cell. In someembodiments, it may be desirable to selectively underglycosylate thesoluble interferon receptors by mutating the potential N-linkedglycosylation sites therein if, e.g., the underglycosylated solubleinterferon receptor exhibits increased activity or contributes toincreased serum half-life. In other embodiments, it may be desirable tounderglycosylate portions of the soluble interferon receptor such thatcertain domains lack N-glycosylation if, for example, such amodification improves the serum half-life of the soluble interferonreceptors. Alternatively, other amino acids in the vicinity ofglycosylation acceptors can be modified, disrupting a recognition motiffor glycosylation enzymes without necessarily changing the amino acidthat would normally be glycosylated.

In some embodiments, glycosylation of a soluble interferon receptor canbe altered by introducing glycosylation sites. For example, the aminoacid sequence of the soluble interferon receptor can be modified tointroduce the consensus sequence for N-linked glycosylation ofAsn-X-Ser/Thr (X is any amino acid other than proline). AdditionalN-linked glycosylation sites can be added anywhere throughout the aminoacid sequence of the soluble interferon receptor. Preferably, theglycosylation sites are introduced in position in the amino acidsequence that does not substantially reduce the activity of the solubleinterferon receptor.

The addition of O-linked glycosylation sites has been reported to alterserum half-life of proteins, such as growth hormone,follicle-stimulating hormone, IGFBP-6, Factor IX, and many others (e.g.,as disclosed in Okada et al., Endocr Rev 2011; 32:2-342; Weenen et al.,J Clin Endocrinol Metab 2004; 89:5204-12; Marinaro et al., EuropeanJournal of Endocrinology 2000; 142:512-6; US 2011/0154516). Accordingly,in some embodiments, O-linked glycosylation (on serine/threonineresidues) of the soluble interferon receptor is altered. Methods foraltering O-linked glycosylation are routine in the art and can beachieved, e.g., by beta-elimination (see, e.g., Huang et al., RapidCommunications in Mass Spectrometry 2002; 16:1199-204; Conrad, CurrProtoc Mol Biol 2001; Chapter 17:Unit17.15A; Fukuda, Curr Protoc MolBiol 2001; Chapter 17; Unit 17.15B; Zachara et al., Curr Protoc Mol Biol2011; Unit 17.6;); by using commercially available kits (e.g.,GlycoProfile™ Beta-Elimination Kit, Sigma); or by subjecting solubleinterferon receptors to treatment with a series of exoglycosidases suchas, but not limited to, β1-4 galactosidase andβ-N-acetylglucosaminidase, until only Gal β1-3GalNAc and/or GlcNAcβ1-3GalNAc remains, followed by treatment with, e.g.,endo-α-N-acetylgalactosaminidase (i.e., O-glycosidase). Such enzymes arecommercially available from, e.g., New England Biolabs. In yet otherembodiments, the soluble interferon receptors are altered to introduceO-linked glycosylation in the soluble interferon receptor as disclosedin, e.g., Okada et al. (supra), Weenen et al. (supra), US2008/0274958;and US2011/0171218. In some embodiments, one or more O-linkedglycosylation consensus sites are introduced into the soluble interferonreceptors, such as CXXGGT/S-C (SEQ ID NO: 29) (van den Steen et al., InCritical Reviews in Biochemistry and Molecular Biology, Michael Cox,ed., 1998; 33:151-208), NST-E/D-A (SEQ ID NO: 30), NITQS (SEQ ID NO:31), QSTQS (SEQ ID NO: 32), D/E-FT-R/K-V (SEQ ID NO: 33), C-E/D-SN (SEQID NO: 34), and GGSC-K/R (SEQ ID NO: 35). Additional O-linkedglycosylation sites can be added anywhere throughout the amino acidsequence of the soluble interferon receptor. Preferably, theglycosylation sites are introduced in position in the amino acidsequence that does not substantially reduce the activity of the solubleinterferon receptors. Alternatively, O-linked sugar moieties areintroduced by chemically modifying an amino acid in the solubleinterferon receptors as described in, e.g., WO 87/05330 and Aplin etal., CRC Crit Rev Biochem 1981; 259-306).

In some embodiments, both N-linked and O-linked glycosylation sites areintroduced into the soluble interferon receptors, preferably inpositions in the amino acid sequence that do not substantially reducethe activity of the soluble interferon receptors.

It is well within the abilities of the skilled artisan to introduce,reduce, or eliminate glycosylation (e.g., N-linked or O-linkedglycosylation) in a soluble interferon receptor and determine usingroutine methods in the art whether such modifications in glycosylationstatus increases or decreases the activity or serum half-life of thesoluble interferon receptor.

In some embodiments, the soluble interferon receptor may comprise analtered glycoform (e.g., an underfucosylated or fucose-free glycan).

In some embodiments, a soluble interferon receptor with alteredglycosylation has a serum half-life that is increased at least about1.5-fold, such as at least 3-fold, at least 5-fold, at least 10-fold, atleast about 20-fold, at least about 50-fold, at least about 100-fold, atleast about 200-fold, at least about 300-fold, at least about 400-fold,at least about 500-fold, at least about 600-fold, at least about700-fold, at least about 800-fold, at least about 900-fold, at leastabout 1000-fold, or 1000-fold or greater relative to the correspondingglycosylated soluble interferon receptors (e.g., a soluble interferonreceptor in which potential N-linked glycosylation sites are notmutated). Routine art-recognized methods can be used to determine theserum half-life of soluble interferon receptors with alteredglycosylation status.

In some embodiments, a soluble interferon receptor with alteredglycosylation (e.g., a aglycosylated, deglycosylated, orunderglycosylated soluble interferon receptors) retains at least 50%,such as at least 60%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, at least 99.5%, or 100% of the activity of the correspondingglycosylated soluble interferon receptor (e.g., a soluble interferonreceptor in which potential N-linked glycosylation sites are notmutated).

In some embodiments, altering the glycosylation status of the solubleinterferon receptors may increase activity, either by directlyincreasing activity, or by increasing bioavailability (e.g., serumhalf-life). Accordingly, in some embodiments, the activity of a solubleinterferon receptor with altered glycosylation is increased by at least1.3-fold, such as at least 1.5-fold, at least 2-fold, at least 2.5-fold,at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold,at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold,at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold,at least 9-fold, at least 9.5 fold, or 10-fold or greater, relative tothe corresponding glycosylated soluble interferon receptor (e.g., asoluble interferon receptor in which potential N-linked glycosylationsites are not mutated).

The skilled artisan can readily determine the glycosylation status ofsoluble interferon receptors using art-recognized methods. In apreferred embodiment, the glycosylation status is determined using massspectrometry. In other embodiments, interactions with Concanavalin A(Con A) can be assessed to determine whether a soluble interferonreceptor is underglycosylated. An underglycosylated soluble interferonreceptor is expected to exhibit reduced binding to Con A-Sepharose whencompared to the corresponding glycosylated soluble interferon receptor.SDS-PAGE analysis can also be used to compare the mobility of anunderglycosylated protein and corresponding glycosylated protein. Theunderglycosylated protein is expected to have a greater mobility inSDS-PAGE compared to the glycosylated protein. Other suitableart-recognized methods for analyzing protein glycosylation status aredisclosed in, e.g., Roth et al., International Journal of CarbohydrateChemistry 2012; 1-10.

Pharmacokinetics, such as serum half-life, of soluble interferonreceptors with different glycosylation status can be assayed usingroutine methods, e.g., by introducing the soluble interferon receptorsin mice, e.g., intravenously, taking blood samples at pre-determinedtime points, and assaying and comparing levels and/or activity of thesoluble interferon receptors in the samples.

Pharmaceutical Compositions

In certain embodiments, a soluble interferon receptor is administeredalone. In certain embodiments, a soluble interferon receptor isadministered prior to the administration of at least one othertherapeutic agent. In certain embodiments, a soluble interferon receptoris administered concurrent with the administration of at least one othertherapeutic agent. In certain embodiments, a soluble interferon receptoris administered subsequent to the administration of at least one othertherapeutic agent. In other embodiments, a soluble interferon receptoris administered prior to the administration of at least one othertherapeutic agent. As will be appreciated by one of skill in the art, insome embodiments, the soluble interferon receptor is combined with theother agent/compound. In some embodiments, the soluble interferonreceptor and other agent are administered concurrently. In someembodiments, the soluble interferon receptor and other agent are notadministered simultaneously, with the soluble interferon receptor beingadministered before or after the agent is administered. In someembodiments, the subject receives both the soluble interferon receptorand the other agent during a same period of prevention, occurrence of adisorder, and/or period of treatment.

Pharmaceutical compositions of the disclosure can be administered incombination therapy, i.e., combined with other agents. In certainembodiments, the combination therapy comprises the soluble interferonreceptor, in combination with at least one other agent. Agents include,but are not limited to, in vitro synthetically prepared chemicalcompositions, antibodies, antigen binding regions, and combinations andconjugates thereof. In certain embodiments, an agent can act as anagonist, antagonist, allosteric modulator, or toxin.

In certain embodiments, the disclosure provides for pharmaceuticalcompositions comprising a soluble interferon receptor together with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant.

In certain embodiments, the invention provides for pharmaceuticalcompositions comprising a soluble interferon receptor and atherapeutically effective amount of at least one additional therapeuticagent, together with a pharmaceutically acceptable diluent, carrier,solubilizer, emulsifier, preservative and/or adjuvant.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Insome embodiments, the formulation material(s) are for s.c. and/or I.V.administration. In certain embodiments, the pharmaceutical compositioncan contain formulation materials for modifying, maintaining orpreserving, for example, the pH, osmolality, viscosity, clarity, color,isotonicity, odor, sterility, stability, rate of dissolution or release,adsorption or penetration of the composition. In certain embodiments,suitable formulation materials include, but are not limited to, aminoacids (such as glycine, glutamine, asparagine, arginine or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite); buffers (such as borate, bicarbonate,Tris-HCl, citrates, phosphates or other organic acids); bulking agents(such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as gelatin); coloring, flavoring and dilutingagents; emulsifying agents; hydrophilic polymers (such aspolyvinylpyrrolidone); low molecular weight polypeptides; salt-formingcounterions (such as sodium); preservatives (such as benzalkoniumchloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol,methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogenperoxide); solvents (such as glycerin, propylene glycol or polyethyleneglycol); sugar alcohols (such as mannitol or sorbitol); suspendingagents; surfactants or wetting agents (such as pluronics, PEG, sorbitanesters, polysorbates such as polysorbate 20, polysorbate 80, triton,tromethamine, lecithin, cholesterol, tyloxapal); stability enhancingagents (such as sucrose or sorbitol); tonicity enhancing agents (such asalkali metal halides, preferably sodium or potassium chloride, mannitolsorbitol); delivery vehicles; diluents; excipients and/or pharmaceuticaladjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A. R.Gennaro, ed., Mack Publishing Company (1995). In some embodiments, theformulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mMNAOAC, pH 5.2, 9% Sucrose.

In certain embodiments, a soluble interferon receptor and/or atherapeutic molecule is linked to a half-life extending vehicle known inthe art. Such vehicles include, but are not limited to, polyethyleneglycol, glycogen (e.g., glycosylation of the soluble interferonreceptor), and dextran. Such vehicles are described, e.g., in U.S.application Ser. No. 09/428,082, now U.S. Pat. No. 6,660,843 andpublished Application No. WO 99/25044.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, Remington's Pharmaceutical Sciences, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantibodies of the invention.

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition can be either aqueous or non-aqueous innature. For example, in certain embodiments, a suitable vehicle orcarrier can be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. In someembodiments, the saline comprises isotonic phosphate-buffered saline. Incertain embodiments, pharmaceutical compositions comprise Tris buffer ofabout pH 7.0-8.5, or acetate buffer of about H 4.0-5.5, which canfurther include sorbitol or a suitable substitute therefore. In certainembodiments, a composition comprising a soluble interferon receptor,with or without at least one additional therapeutic agents, can beprepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, in certain embodiments, a compositioncomprising a soluble interferon receptor, with or without at least oneadditional therapeutic agent, can be formulated as a lyophilizate usingappropriate excipients such as sucrose.

In certain embodiments, the pharmaceutical composition can be selectedfor parenteral delivery. In certain embodiments, the compositions can beselected for inhalation or for delivery through the digestive tract,such as orally. The preparation of such pharmaceutically acceptablecompositions is within the ability of one skilled in the art.

In certain embodiments, the formulation components are present inconcentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated,a therapeutic composition can be in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising a desired a solubleinterferon receptor, with or without additional therapeutic agents, in apharmaceutically acceptable vehicle. In certain embodiments, a vehiclefor parenteral injection is sterile distilled water in which solubleinterferon receptor, with or without at least one additional therapeuticagent, is formulated as a sterile, isotonic solution, properlypreserved. In certain embodiments, the preparation can involve theformulation of the desired molecule with an agent, such as injectablemicrospheres, bio-erodible particles, polymeric compounds (such aspolylactic acid or polyglycolic acid), beads or liposomes, that canprovide for the controlled or sustained release of the product which canthen be delivered via a depot injection. In certain embodiments,hyaluronic acid can also be used, and can have the effect of promotingsustained duration in the circulation. In certain embodiments,implantable drug delivery devices can be used to introduce the desiredmolecule.

In certain embodiments, a pharmaceutical composition can be formulatedfor inhalation. In certain embodiments, a soluble interferon receptor,with or without at least one additional therapeutic agent, can beformulated as a dry powder for inhalation. In certain embodiments, aninhalation solution comprising a soluble interferon receptor, with orwithout at least one additional therapeutic agent, can be formulatedwith a propellant for aerosol delivery. In certain embodiments,solutions can be nebulized. Pulmonary administration is furtherdescribed in PCT application no. PCT/US94/001875, which describespulmonary delivery of chemically modified proteins.

In certain embodiments, it is contemplated that formulations can beadministered orally. In certain embodiments, a soluble interferonreceptor, with or without at least one additional therapeutic agents,that is administered in this fashion can be formulated with or withoutthose carriers customarily used in the compounding of solid dosage formssuch as tablets and capsules. In certain embodiments, a capsule can bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. In certain embodiments, at leastone additional agent can be included to facilitate absorption of asoluble interferon receptor and/or any additional therapeutic agents. Incertain embodiments, diluents, flavorings, low melting point waxes,vegetable oils, lubricants, suspending agents, tablet disintegratingagents, and binders can also be employed.

In certain embodiments, a pharmaceutical composition can involve aneffective quantity of a soluble interferon receptor, with or without atleast one additional therapeutic agents, in a mixture with non-toxicexcipients which are suitable for the manufacture of tablets. In certainembodiments, by dissolving the tablets in sterile water, or anotherappropriate vehicle, solutions can be prepared in unit-dose form. Incertain embodiments, suitable excipients include, but are not limitedto, inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving a soluble interferonreceptor, with or without at least one additional therapeutic agent(s),in sustained- or controlled-delivery formulations. In certainembodiments, techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See for example, PCT Application No.PCT/US93/00829 which describes the controlled release of porouspolymeric microparticles for the delivery of pharmaceuticalcompositions. In certain embodiments, sustained-release preparations caninclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices can includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et al, Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J Biomed Mater Res, 15:167-277 (1981) and Langer, Chem Tech, 12:98-105 (1982)), ethylene vinylacetate (Langer et al, supra) or poly-D(−)-3-hydroxybutyric acid (EP133,988). In certain embodiments, sustained release compositions canalso include liposomes, which can be prepared by any of several methodsknown in the art. See, e.g., Eppstein et al, PNAS, 82:3688-3692 (1985);EP 036,676; EP 088,046 and EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. In certain embodiments, this can be accomplishedby filtration through sterile filtration membranes. In certainembodiments, where the composition is lyophilized, sterilization usingthis method can be conducted either prior to or following lyophilizationand reconstitution. In certain embodiments, the composition forparenteral administration can be stored in lyophilized form or in asolution. In certain embodiments, parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

In certain embodiments, once the pharmaceutical composition has beenformulated, it can be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Incertain embodiments, such formulations can be stored either in aready-to-use form or in a form (e.g., lyophilized) that is reconstitutedprior to administration.

In certain embodiments, kits are provided for producing a single-doseadministration unit. In certain embodiments, the kit can contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are included.

In certain embodiments, the effective amount of a pharmaceuticalcomposition comprising a soluble interferon receptor, with or without atleast one additional therapeutic agent, to be employed therapeuticallywill depend, for example, upon the therapeutic context and objectives.One skilled in the art will appreciate that the appropriate dosagelevels for treatment, according to certain embodiments, will thus varydepending, in part, upon the molecule delivered, the indication forwhich a soluble interferon receptor, with or without at least oneadditional therapeutic agent, is being used, the route ofadministration, and the size (body weight, body surface or organ size)and/or condition (the age and general health) of the patient. In certainembodiments, the clinician can titer the dosage and modify the route ofadministration to obtain the optimal therapeutic effect. In certainembodiments, a typical dosage can range from about 0.1 μg/kg to up toabout 100 mg/kg or more, depending on the factors mentioned above. Incertain embodiments, the dosage can range from 0.1 μg/kg up to about 100mg/kg; or 1 μg/kg up to about 100 mg/kg; or 5 μg/kg up to about 100mg/kg.

In certain embodiments, the frequency of dosing will take into accountthe pharmacokinetic parameters of a soluble interferon receptor and/orany additional therapeutic agents in the formulation used. In certainembodiments, a clinician will administer the composition until a dosageis reached that achieves the desired effect. In certain embodiments, thecomposition can therefore be administered as a single dose, or as two ormore doses (which may or may not contain the same amount of the desiredmolecule) over time, or as a continuous infusion via an implantationdevice or catheter. Further refinement of the appropriate dosage isroutinely made by those of ordinary skill in the art and is within theambit of tasks routinely performed by them. In certain embodiments,appropriate dosages can be ascertained through use of appropriatedose-response data.

In certain embodiments, the route of administration of thepharmaceutical composition is in accord with known methods, e.g. orally,through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,subcutaneously, intra-ocular, intraarterial, intraportal, orintralesional routes; by sustained release systems or by implantationdevices. In certain embodiments, the compositions can be administered bybolus injection or continuously by infusion, or by implantation device.

In certain embodiments, the composition can be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated. In certainembodiments, where an implantation device is used, the device can beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule can be via diffusion, timed-release bolus, or continuousadministration.

In certain embodiments, it can be desirable to use a pharmaceuticalcomposition comprising a soluble interferon receptor, with or without atleast one additional therapeutic agent, in an ex vivo manner. In suchinstances, cells, tissues and/or organs that have been removed from thepatient are exposed to a pharmaceutical composition comprising a solubleinterferon receptor, with or without at least one additional therapeuticagent, after which the cells, tissues and/or organs are subsequentlyimplanted back into the patient.

In certain embodiments, a soluble interferon receptor and/or anyadditional therapeutic agents can be delivered by implanting certaincells that have been genetically engineered, using methods such as thosedescribed herein, to express and secrete the polypeptides. In certainembodiments, such cells can be animal or human cells, and can beautologous, heterologous, or xenogeneic. In certain embodiments, thecells can be immortalized. In certain embodiments, in order to decreasethe chance of an immunological response, the cells can be encapsulatedto avoid infiltration of surrounding tissues. In certain embodiments,the encapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

In Vitro Assays

Various in vitro assays known in the art can be used to assess theefficacy of the soluble interferon receptors of the invention.

For example, soluble interferon receptors can be assessed for theirability to inhibit IFN-α and/or IFN-β induced secreted alkalinephosphatase (SEAP) production in HEK-Blue α/β cells. HEK-Blue IFN-α/βcells (Invivogen, Catalog #hkb-ifnab) produce and secrete SEAP inresponse to stimulation by IFN-α or IFN-β.

To assess the ability of the soluble interferon receptors to inhibitIFN-α and/or IFN-β activity, IFNα and/or IFN-β can be added toinhibitors (e.g., soluble interferon receptors (e.g., RSLV-601-604,RSLV-602-603) as well as control molecules such as anti-IFN-α,anti-IFN-β, and human IgG) in a tissue culture plate. HEK-Blue IFN-α/βcells can then be added to the plate and incubated for a predeterminedamount of time at 37° C. To assess SEAP activity, cell supernatant canthen be added to QUANTI-Blue reagent and incubated for a predeterminedamount of time at 37° C. SEAP activity can be detected by measuringabsorbance at 620 nm.

The effectiveness of the soluble interferon receptors is demonstrated bycomparing the results of an assay from cells treated with the solubleinterferon receptors disclosed herein to the results of the assay fromcells treated with a control. After treatment with an effective solubleinterferon receptor, the levels of IFN-α and/or IFN-β are generallycomparable to the levels measured following treatment with an anti-IFN-αor anti-IFN-(3 control. After treatment with an effective solubleinterferon receptor, the levels of IFN-α and/or IFN-β are generallyreduced relative to the levels measured following treatment with anegative control such as human IgG.

Methods of Treatment

The soluble interferon receptors of the disclosure are particularlyeffective in the treatment of autoimmune disorders or abnormal immuneresponses. In this regard, it will be appreciated that the solubleinterferon receptors of the present disclosure may be used to control,suppress, modulate, treat, or eliminate dysregulated immune responsesresulting from excess production of interferon.

In another aspect, a soluble interferon receptors is adapted forpreventing (prophylactic) or treating (therapeutic) a disease ordisorder, such as an autoimmune disease, in a mammal by administering asoluble interferon receptor in a therapeutically effective amount or asufficient amount to the mammal in need thereof, wherein the disease isprevented or treated. Any route of administration suitable for achievingthe desired effect is contemplated by the invention (e.g., intravenous,intramuscular, subcutaneous). Treatment of the disease condition mayresult in a decrease in the symptoms associated with the condition,which may be long-term or short-term, or even a transient beneficialeffect.

Numerous disease conditions are suitable for treatment with the solubleinterferon receptors of the disclosure. For example, in some aspects,the disease or disorder is an autoimmune disease or cancer. In some suchaspects, the autoimmune disease is insulin-dependent diabetes mellitus,multiple sclerosis, experimental autoimmune encephalomyelitis,rheumatoid arthritis, experimental autoimmune arthritis, myastheniagravis, thyroiditis, an experimental form of uveoretinitis, Hashimoto'sthyroiditis, primary myxoedema, thyrotoxicosis, pernicious anaemia,autoimmune atrophic gastritis, Addison's disease, premature menopause,male infertility, juvenile diabetes, Goodpasture's syndrome, pemphigusvulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis,autoimmune haemolytic anaemia, idiopathic leucopenia, primary biliarycirrhosis, active chronic hepatitis Hbs-ve, cryptogenic cirrhosis,ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener'sgranulomatosis, polymyositis, dermatomyositis, discoid LE, SLE, orconnective tissue disease.

In a specific embodiment, a soluble interferon receptor is used toprevent or treat SLE or Sjogren's syndrome. The effectiveness of asoluble interferon receptor is demonstrated by comparing the level ofexpression of certain known IFN regulated genes in mammals treated witha soluble interferon receptor disclosed herein to mammals treated withcontrol formulations. In some embodiments, the expression level of one,two, three, four, five or more IFN regulated genes is measured. Forexample, in some embodiments, the expression level of three or more IFNregulated genes (e.g., HERC5, EPSTI, CMPK2) is measured. In someembodiments the IFN regulated genes include those described by Bennettet al., J. Exp. Med., Vol. 197, No. 6, 711-723, March 2003. and Kennedyet al. Lupus Science and Medicine, 2015; 2:e00080.Doi:10.1136/lupus-2014-000080, both of which are incorporated herein byreference.

For example, a human subject in need of treatment is selected oridentified (e.g., a patient who fulfills the American College ofRheumatology criteria for SLE, or a patient who fulfills theAmerican-European Consensus Sjogren's Classification Criteria). Thesubject can be in need of, e.g., reducing a cause or symptom of SLE orSjogren's syndrome. The identification of the subject can occur in aclinical setting, or elsewhere, e.g., in the subject's home through thesubject's own use of a self-testing kit.

At time zero, a suitable first dose of a soluble interferon receptor isadministered to the subject. The soluble interferon receptor isformulated as described herein. After a period of time following thefirst dose, e.g., 7 days, 14 days, and 21 days, the subject's conditionis evaluated, e.g., by measuring IFN regulated gene expression. Forexample, the expression of one or more of HERC5, EPSTI, and CMPK2, whichare interferon stimulated genes, can be assessed. Other relevantcriteria can also be measured. The number and strength of doses areadjusted according to the subject's needs. The progress of treatment maybe monitored by assaying for a change in IFN regulated gene expression.After treatment, a decrease and/or improvement can be noted in theexpression of the subject's IFN regulated gene expression relative tothe IFN regulated gene expression prior to the treatment, or relative tothe levels measured in a similarly afflicted but untreated/controlsubject. For example, the expression of HERC5, EPSTI, and/or CMPK2,which are interferon stimulated genes, would be decreased relative tothe expression of these three genes prior to treatment, or relative tothe levels of these genes in a similarly afflicted by untreated/controlsubject.

In some embodiments, the IFN regulated gene expression is measured bydrawing whole blood from a subject, extracting the RNA and analyzing theexpression of the interferon regulated genes (for example, HERC5, EPSTI,and/or CMPK2) by using techniques well-known in the art, such as PCR.Methods for assaying for IFN regulated gene expression are described inKennedy et al. Lupus Science and Medicine, 2015; 2:e00080.Doi:10.1136/lupus-2014-000080 and Furie et al., Arthritis &Rheumatology, Vo. 69, No. 2, February 2017, 376-386, both of which areincorporated herein by reference.

In another example, a rodent subject in need of treatment is selected oridentified. The identification of the subject can occur in a laboratorysetting or elsewhere. At time zero, a suitable first dose of a solubleinterferon receptor is administered to the subject. The a solubleinterferon receptor is formulated as described herein. After a period oftime following the first dose, e.g., 7 days, 14 days, and 21 days, thesubject's condition is evaluated, e.g., by measuring IFN regulated geneexpression. Other relevant criteria can also be measured. The number andstrength of doses are adjusted according to the subject's needs. Theprogress of treatment may be monitored by assaying for a change in IFNregulated gene expression.

After treatment, the subject's IFN regulated gene expression are loweredand/or improved relative to the IFN regulated gene expression prior tothe treatment, or relative to the levels measured in a similarlyafflicted but untreated/control subject.

In some embodiments, IFN regulated genes that may be upregulated in anautoimmune disorder (e.g., SLE) include, IFP35 IFN inducible, IRF7B,MX1, MX2, XIAP ass. factor, GS3686, P69 2′5′ oligoA synthetase, hep-Cass. microtubular agg. prot., RIGE/TSA1 sim to mouse Ly6, agrin prec,IFI-56 IFN inducible, EST sim. to IFN ind 17 kD protein, cig 5, ISG 15,TRIP 14 2′5′ oligoA synthetase-like, cig49, MCP-1 monocytechemoattractant, Tudor rpt ass with PCTAIRE, MMTRA 1B phospholipidscramblase, FACL 1 fatty acid coenzyme-A ligase, TRAIL, 2′5′ oligoAsynthetase E18 isoform, GBP-1 guanylate binding protein 1, C1-INH CC1inhibitor, CD64 rec for Fc fragment of IgG, C2 complement component,hPD-ECGF end. platelet der. GF1, ISGF3, EST hute1, TSC403 DC LAMP,MAC2-BP scavenger receptor, 1-8U, TAP1, IFI 6-16, Novel phorbolin-likegene, G6PD guanosine monoP reductase, HERC5, EPSTI, and CMPK2.

In some embodiments, IFN regulated genes that may be downregulated in anautoimmune disorder (e.g., SLE) include TCRγ T-cell receptor delta, LEU1 leukemia ass. gene1, COX11P cyt C oxidase ass. prot, JKTBP nucribonucleoprotein D-like, TPRD tetra tricopeptide rpt, DAP3 death ass.protein, mRNA U90916, PRIP prion protein, ANT 3 ADP.ATP translocase,E1F-4B transl.initiation fac, PABP4 polyA binding protein, RAB 4A GTPbinding protein, and CD3γ.

In some embodiments, the effectiveness of a soluble interferon receptoris demonstrated by assessing the Cutaneous Lupus Erythematosus DiseaseArea and Severity Index (CLASI), British Isles Lupus Assessment Group(BILAG) index, Systemic Lupus Erythematosus (SLE) Responder Index(SRI-4), and/or the Functional Assessment of Chronic Illness Therapy(FACIT) fatigue scale in mammals treated with a soluble interferonreceptor disclosed herein when compared to mammals treated with controlformulations. In some embodiments, a mammal treated with a solubleinterferon receptor will demonstrate an improvement in the CLASIseverity index, BILAG index, SRI-4 index, and/or the FACIT fatigue scalewhen compared to the mammal's CLASI severity index, BILAG index, SRI-4index, and/or the FACIT fatigue scale prior to the treatment, or whencompared to a mammal treated with a control formulation.

In some embodiments, the effectiveness of a soluble interferon receptoris demonstrated by assessing a reduction in steroid use in mammalstreated with a soluble interferon receptor disclosed herein whencompared to mammals treated with control formulations. In someembodiments, a mammal treated with a soluble interferon receptor willdemonstrate a reduction in steroid use when compared to the mammal'ssteroid use prior to the treatment, or when compared to a mammal treatedwith a control formulation.

For example, a human subject in need of treatment is selected oridentified (e.g., a patient who fulfills the American College ofRheumatology criteria for SLE, or a patient who fulfills theAmerican-European Consensus Sjogren's Classification Criteria). Thesubject can be in need of, e.g., reducing a cause or symptom of SLE orSjogren's syndrome. The identification of the subject can occur in aclinical setting, or elsewhere, e.g., in the subject's home through thesubject's own use of a self-testing kit.

At time zero, a suitable first dose of a soluble interferon receptor isadministered to the subject. The soluble interferon receptor isformulated as described herein. After a period of time following thefirst dose, e.g., 7 days, 14 days, and 21 days, the subject's conditionis evaluated, e.g., by CLASI severity index, BILAG index, SRI-4 index,the FACIT fatigue scale, and/or a reduction in steroid use. Otherrelevant criteria can also be measured. The number and strength of dosesare adjusted according to the subject's needs. After treatment, animprovement in one or more of the following outcomes can be noted: (1)an improvement in the CLASI severity index relative to the CLASIseverity index prior to treatment, or relative to a similarly afflictedbut untreated/control subject, (2) an improvement in the BILAG indexrelative to the BILAG index prior to treatment, or relative to asimilarly afflicted but untreated/control subject, (3) an improvement inthe SRI-4 index relative to the SRI-4 index prior to treatment, orrelative to a similarly afflicted but untreated/control subject, (4) animprovement can be noted in the FACIT fatigue scale relative to theFACIT fatigue scale prior to treatment, or relative to a similarlyafflicted but untreated/control subject, (5) a reduction in steroid userelative to steroid use prior to treatment, or relative to a similarlyaffected but untreated/control subject.

In another example, a rodent subject in need of treatment is selected oridentified. The identification of the subject can occur in a laboratorysetting or elsewhere. At time zero, a suitable first dose of a solubleinterferon receptor is administered to the subject. The a solubleinterferon receptor is formulated as described herein. After a period oftime following the first dose, e.g., 7 days, 14 days, and 21 days, thesubject's condition is evaluated, e.g., by CLASI severity index, BILAGindex, SRI-4 index, the FACIT fatigue scale, and/or a reduction insteroid use. Other relevant criteria can also be measured. The numberand strength of doses are adjusted according to the subject's needs.

After treatment, an improvement in one or more of the following outcomescan be noted: (1) an improvement in the CLASI severity index relative tothe CLASI severity index prior to treatment, or relative to a similarlyafflicted but untreated/control subject, (2) an improvement in the BILAGindex relative to the BILAG index prior to treatment, or relative to asimilarly afflicted but untreated/control subject, (3) an improvement inthe SRI-4 index relative to the SRI-4 index prior to treatment, orrelative to a similarly afflicted but untreated/control subject, (4) animprovement can be noted in the FACIT fatigue scale relative to theFACIT fatigue scale prior to treatment, or relative to a similarlyafflicted but untreated/control subject, (5) a reduction in steroid userelative to steroid use prior to treatment, or relative to a similarlyaffected but untreated/control subject.

Another aspect of the present invention is to use gene therapy methodsfor treating or preventing disorders, diseases, and conditions with oneor more soluble interferon receptors. The gene therapy methods relate tothe introduction of interferon receptor Fc construct nucleic acid (DNA,RNA and antisense DNA or RNA) sequences into an animal in need thereofto achieve expression of the polypeptide or polypeptides of the presentdisclosure. This method can include introduction of one or morepolynucleotides encoding a interferon receptor Fc construct of thepresent disclosure operably coupled to a promoter and any other geneticelements necessary for the expression of the polypeptide by the targettissue.

In gene therapy applications, interferon receptor Fc construct genes areintroduced into cells in order to achieve in vivo synthesis of atherapeutically effective genetic product. “Gene therapy” includes bothconventional gene therapies where a lasting effect is achieved by asingle treatment, and the administration of gene therapeutic agents,which involves the one time or repeated administration of atherapeutically effective DNA or mRNA. The oligonucleotides can bemodified to enhance their uptake, e.g., by substituting their negativelycharged phosphodiester groups by uncharged groups.

OTHER EMBODIMENTS

The disclosure also relates to the following embodiments which featureheterodimers of the disclosure and use thereof. Throughout this section,the term embodiment is abbreviated as ‘E’ followed by an ordinal. Forexample, E1 is equivalent to Embodiment 1.

E1. A heterodimer comprising a first polypeptide and a secondpolypeptide, wherein the first polypeptide comprises an interferonreceptor 1 (IFNAR1) domain operatively coupled with or without a linkerdomain to a variant Fc domain, and wherein the second polypeptidecomprises an interferon receptor 2 (IFNAR2) domain operatively coupledwith or without a linker domain to a variant Fc domain.E2. The heterodimer of claim E1, wherein the variant Fc domain of thefirst or second polypeptide comprises one or more amino acidsubstitutions which increase the formation of heterodimers as comparedto wild-type.E3. The heterodimer of claim E2, wherein the variant Fc domain of thefirst or second polypeptide comprises one or more amino acidsubstitutions selected from the group consisting of T350V, L351Y, F405A,and Y407V.E4. The heterodimer of claim E2, wherein the variant Fc domain of thefirst or second polypeptide comprises one or more amino acidsubstitutions selected from the group consisting of T350V, T366L, K392L,and T394W.E5. The heterodimer of claim E1, wherein the Fc domain of the first orsecond polypeptide comprises a human immunoglobulin Fc domain, such as ahuman IgG1 Fc domain.E6. The heterodimer of claim E5, wherein the Fc domain of the first orsecond polypeptide comprises a hinge domain, a CH2 domain and a CH3domain.E7. The heterodimer of claim E5, wherein the Fc domain comprises anamino acid sequence having one or more of the amino acid substitutionsP238S, P331S, SCC, SSS (residues 220, 226, and 229), G236R, L328R,L234A, and L235A.E8. The heterodimer of claim E1, wherein the variant Fc domain of thefirst or second polypeptide further comprises one or more amino acidsubstitutions selected from the group consisting of C220S, P238S, andP331S.E9. The heterodimer of any one of claims E1-E4, wherein the variant Fcdomain further comprises one or more amino acid substitutions selectedfrom the group consisting of C220S, P238S, and P331S.E 10. The heterodimer of claim E1, wherein the interferon receptor 1(IFNAR1) domain of the first polypeptide is operatively coupled to thevariant Fc domain via a linker domain.E11. The heterodimer of claim E1, wherein the interferon receptor 2(IFNAR2) domain of the second polypeptide is operatively coupled to thevariant Fc domain via a linker domain.E12. The heterodimer of claim E10 or claim E11, wherein the linkerdomain is a polypeptide linker.E13. The heterodimer of claim E12, wherein the linker domain is aGly-Ser linker.E14. The heterodimer of claim E1, wherein the variant Fc domain of thefirst polypeptide is different than the variant Fc domain of the secondpolypeptide.E15. The heterodimer of claim E14, wherein the variant Fc domain of thefirst polypeptide comprises one or more amino acid substitutionsselected from the group consisting of T350V, L351Y, F405A, and Y407V,and wherein the variant Fc domain of the second polypeptide comprisesone or more amino acid substitutions selected from the group consistingof T350V, T366L, K392L, and T394W.E16. The heterodimer of claim E14, wherein the variant Fc domain of thefirst polypeptide comprises one or more amino acid substitutionsselected from the group consisting of T350V, T366L, K392L, and T394W,and wherein the variant Fc domain of the second polypeptide comprisesone or more amino acid substitutions selected from the group consistingof T350V, L351Y, F405A, and Y407V.E17. The heterodimer of any one claims E15 or E16, wherein the variantFc domain of the first polypeptide further comprises one or more aminoacid substitutions selected from the group consisting of C220S, P238S,and P331S, and wherein the variant Fc domain of the second polypeptidefurther comprises one or more amino acid substitutions selected from thegroup consisting of C220S, P238S, and P331S.E18. A heterodimer comprising a first polypeptide and a secondpolypeptide,

wherein the first polypeptide comprises an interferon receptor 2(IFNAR2) domain operatively coupled with or without a linker domain to avariant Fc domain comprising one or more amino acid substitutionsselected from the group consisting of T350V, L351Y, F405A, and Y407V,and

wherein the second polypeptide comprises an interferon receptor 1(IFNAR1) domain operatively coupled with or without a linker domain to avariant Fc domain comprising one or more amino acid substitutionsselected from the group consisting of T350V, T366L, K392L, and T394W.

E19. A heterodimer comprising a first polypeptide and a secondpolypeptide,

wherein the first polypeptide comprises an interferon receptor 1(IFNAR1) domain operatively coupled with or without a linker domain to avariant Fc domain comprising one or more amino acid substitutionsselected from the group consisting of T350V, L351Y, F405A, and Y407V,and

wherein the second polypeptide comprises an interferon receptor 2(IFNAR2) domain operatively coupled with or without a linker domain to avariant Fc domain comprising one or more amino acid substitutionsselected from the group consisting of T350V, T366L, K392L, and T394W.

E20. The heterodimer of any one claims E18-E19, wherein the variant Fcdomain of the first polypeptide further comprises one or more mutationselected from the group consisting of C220S, P238S, and P331S, andwherein the variant Fc domain of the second polypeptide furthercomprises one or more mutation selected from the group consisting ofC220S, P238S, and P331S.E21. A heterodimer comprising

a first polypeptide and a second polypeptide,

wherein the first polypeptide comprises an interferon receptor 2(IFNAR2) domain operatively coupled via a Gly-Ser linker domain to avariant Fc domain, wherein the variant Fc domain of the firstpolypeptide comprises one or more amino acid substitutions selected fromthe group consisting of T350V, L351Y, F405A, and Y407V, and wherein thevariant Fc domain of the first polypeptide further comprises one or moremutation selected from the group consisting of C220S, P238S, and P331S;and

wherein the second polypeptide comprises an interferon receptor 1(IFNAR1) operatively coupled via a Gly-Ser linker domain to a variant Fcdomain, wherein the variant Fc domain of the second polypeptidecomprises one or more amino acid substitutions selected from the groupconsisting of T350V, T366L, K392L, and T394W, and wherein the variant Fcdomain of the second polypeptide further comprises one or more mutationselected from the group consisting of C220S, P238S, and P331S.

E22. A heterodimer comprising

a first polypeptide and a second polypeptide,

wherein the first polypeptide comprises an interferon receptor 1(IFNAR1) domain operatively coupled via a Gly-Ser linker domain to avariant Fc domain, wherein the variant Fc domain of the firstpolypeptide comprises one or more amino acid substitutions selected fromthe group consisting of T350V, L351Y, F405A, and Y407V, and wherein thevariant Fc domain of the first polypeptide further comprises one or moremutation selected from the group consisting of C220S, P238S, and P331S;and

wherein the second polypeptide comprises an interferon receptor 2(IFNAR2) operatively coupled via a Gly-Ser linker domain to a variant Fcdomain, wherein the variant Fc domain of the second polypeptidecomprises one or more amino acid substitutions selected from the groupconsisting of T350V, T366L, K392L, and T394W, and wherein the variant Fcdomain of the second polypeptide further comprises one or more mutationselected from the group consisting of C220S, P238S, and P331S.

E23. A composition comprising the heterodimer of any of the precedingclaims and a pharmaceutically acceptable carrier.E24. A nucleic acid encoding the first polypeptide of the heterodimeraccording to claim E1.E25. A nucleic acid encoding the second polypeptide of the heterodimeraccording to claim E1.E26. A recombinant expression vector comprising a nucleic acid accordingto claim E24.E27. A recombinant expression vector comprising a nucleic acid accordingto claim E25.E28. A host cell transformed with the recombinant expression vector ofclaim E26 and the recombinant expression vector of claim E27.E29. A method of making the heterodimer of claim E1, comprising:providing a host cell comprising a nucleic acid sequence that encodesthe first polypeptide and a nucleic acid that encodes the secondpolypeptide; and maintaining the host cell under conditions in which thefirst and second polypeptides are expressed.E30. The heterodimer of claim E1 for use in a method for treating orpreventing a condition associated with an abnormal immune response.E31. The heterodimer of claim E30, wherein the condition is anautoimmune disease.E32. The heterodimer of claim E31, wherein the autoimmune disease isSLE.E33. The heterodimer of claim E1 for use in the manufacture of amedicament for treating or preventing a condition associated with anabnormal immune response.E34. The heterodimer of claim E33, wherein the condition is anautoimmune disease.E35. The heterodimer of claim E34, wherein the autoimmune disease isSLE.E36. The heterodimer of claim E1, wherein the heterodimer binds type Iinterferons.E37. The heterodimer of claim E36, wherein the type I interferon isinterferon α.E38. The heterodimer of claim E36, wherein the type I interferon isinterferon β.E39. The heterodimer of claim E1, wherein the heterodimer bindsinterferon α to a similar extent as a control anti-IFNα antibody.E40. The heterodimer of claim E1, wherein the heterodimer bindsinterferon β to a similar extent as a control anti-IFNβ antibody.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W. H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al, Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S.Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Example 1 Generating Soluble Interferon Receptors

Various embodiments of the soluble interferon receptors are shown inFIG. 1 with amino acid sequences of each presented in Table 1. Thefollowing interferon receptor extracellular domain (ECD)-immunoglobulinFc fusion proteins were constructed: RSLV-601, RSLV-602, RSLV-603,RSLV-604, RSLV-606, RSLV-608, RSLV-611, and RSLV-613 (FIG. 1).Constructs were generated through direct synthesis using commerciallyavailable services. Amino acid sequences were provided to GeneArt; andcodon utilization was optimized for and the genes were synthesized andinserted into the mammalian cell expression vector pcDNA3.1±

RSLV-601 (SEQ ID NO:1) has the configuration: Leader Sequence(MDWTWRILFLVAAATGTHA)-IFNAR1 ECD-(Gly₄Ser)₄-Fc domain (216-447) withmutations C220S/P238S/P331S/T350V/T366L/K392L/T394W, wherein the IFNAR1ECD is operably coupled via a (Gly₄Ser)₄ sequence to an Fc domain(216-447) with mutations C220S/P238S/P331S/T350V/T366L/K392L/T394W.

RSLV-602 (SEQ ID NO:2) has the configuration: Leader Sequence(MDWTWRILFLVAAATGTHA)-IFNAR2 ECD-(Gly₄Ser)₄-Fc domain (216-447) withmutations C220S/P238S/P331S/T350V/T366L/K392L/T394W, wherein IFNAR2 ECDis operably coupled via a (Gly₄Ser)₄ sequence to an Fc domain (216-447)with mutations C220S/P238S/P331S/T350V/T366L/K392L/T394W.

RSLV-603 (SEQ ID NO:3) has the configuration: Leader Sequence(MDWTWRILFLVAAATGTHA)-IFNAR1 ECD-(Gly₄Ser)₄-Fc domain (216-447) withmutations C220S/P238S/P331S/T350V/L351Y/F405A/Y407V, wherein the IFNAR1ECD is operably coupled via a (Gly₄Ser)₄ sequence to an Fc domain(216-447) with mutations C220S/P238S/P331S/T350V/L351Y/F405A/Y407V.

RSLV-604 (SEQ ID NO:4) has the configuration: Leader Sequence(MDWTWRILFLVAAATGTHA)-IFNAR2 ECD-(Gly₄Ser)₄-Fc domain (216-447) withmutations C220S/P238S/P331S/T350V/L351Y/F405A/Y407V, wherein the IFNAR2ECD is operably coupled via a (Gly₄Ser)₄ sequence to an Fc domain(216-447) with mutations C220S/P238S/P331S/T350V/L351Y/F405A/Y407V.

RSLV-606 (SEQ ID NO: 52) has the configuration: Leader Sequence(MDWTWRILFLVAAATGTHA)-IFNAR1 ECD-(Gly₄Ser)₂-Fc domain (216-447) withmutations C220S/P238S/P331S/T350V/T366L/K392L/T394W, wherein the IFNAR1ECD is operably coupled via a (Gly₄Ser)₂ sequence to an Fc domain(216-447) with mutations C220S/P238S/P331S/T350V/T366L/K392L/T394W.

RSLV-608 (SEQ ID NO: 56) has the configuration: Leader Sequence(MDWTWRILFLVAAATGTHA)-IFNAR1 ECD-Fc domain (216-447) with mutationsC220S/P238S/P331S/T350V/T366L/K392L/T394W, wherein the IFNAR1 ECD isoperably coupled to an Fc domain (216-447) with mutationsC220S/P238S/P331S/T350V/T366L/K392L/T394W.

RSLV-611 (SEQ ID NO: 54) has the configuration: Leader Sequence(MDWTWRILFLVAAATGTHA)-IFNAR2 ECD-(Gly₄Ser)₂-Fc domain (216-447) withmutations C220S/P238S/P331S/T350V/L351Y/F405A/Y407V, wherein the IFNAR2ECD is operably coupled via a (Gly₄Ser)₂ sequence to an Fc domain(216-447) with mutations C220S/P238S/P331S/T350V/L351Y/F405A/Y407V.

RSLV-613 (SEQ ID NO:58) has the configuration: Leader Sequence(MDWTWRILFLVAAATGTHA)-IFNAR2 ECD-Fc domain (216-447) with mutationsC220S/P238S/P331S/T350V/L351Y/F405A/Y407V, wherein the IFNAR2 ECD isoperably coupled via a (Gly₄Ser) sequence to an Fc domain (216-447) withmutations C220S/P238S/P331S/T350V/L351Y/F405A/Y407V.

The interferon receptor Fc constructs of the invention can also begenerated using conventional cloning techniques well-known in the art,for example, by preparing modular cassettes of each component of theinterferon receptor Fc constructs (e.g., INFAR ECD, linker domain, Fc)with compatible restriction enzyme sites to allow for shuttling anddomain swapping. A polynucleotide encoding each component of theinterferon receptor Fc constructs (e.g., INFAR ECD, linker domain,immunoglobulin Fc) can be readily obtained by amplifying the componentof interest using polymerase chain reaction (PCR) from an appropriatecDNA library. For example, the full length nucleotide sequences of humanINFAR ECD, linker domain, and immunoglobulin Fc can be amplified fromrandom primed and oligo dT primed cDNA derived from commerciallyavailable human pancreatic total RNA using sequence specific 5′ and 3′primers based on published sequences of the component being amplified.

Linkers (e.g., (Gly₄Ser)₄) linkers can be generated by overlap PCR usingroutine methods, or through direct synthesis using commerciallyavailable services, and designed to have overhangs or be blunt tofacilitate subsequent cloning to allow for fusion with other domains ofinterest.

Example 2 Transient Expression of Soluble Interferon Receptors

For transient expression, pairs of expression vectors containing theinterferon receptor ECD-Fc constructs from Example 1 were co-transfectedinto CHO-S cells. For example, RSLV-601 and RSLV-604; RSLV-602 andRSLV-603; RSLV-606 and RSLV-611; and RSLV-608 and RSLV-613 wereco-transfected into CHO-S cells.

Plasmids obtained from GeneArt were transformed into DH10B competent E.coli and the cultures expanded under ampicillin selection. Plasmid DNAsubsequently was isolated from the cultures using QIAGEN plasmid plusmaxi kits.

Transfections were performed using the FreeStyle MAX CHO ExpressionSystem obtained from Life Technologies. One day prior to transfection,CHO-S cells were seeded at a density of 5×10⁵ cells/ml in 100 ml ofFreeStyle CHO expression medium supplemented with 8 mM L-glutamine; theflasks subsequently were placed on an orbital shaker rotating at 120-135rpm and incubated overnight in an 8% CO₂ incubator at 37° C. On the dayof transfection, the CHO-S cells were harvested then re-seeded at adensity of 1×10⁶ cells/ml in 100 ml of FreeStyle CHO Expression Mediumsupplemented with 8 mM L-glutamine. 1:1 co-transfections were performed.62.5 μg of RSLV-601 and 62.5 μg of RSLV-604; and 62.5 μg of RSLV-602 and62.5 μg or RSLV-603 were added into OptiPRO SFM with a final volume of 2ml, and mixed by repeated inversion. In a separate tube, 125μl ofFreeStyle MAX transfection reagent was mixed with 1875 μl of OptiPRO SFMand mixed by repeated inversion. The diluted FreeStyle MAX transfectionreagent then was immediately added to the diluted plasmid DNA solution(total volume=4 ml); the resulting solution was mixed gently byinversion and complexes were allowed to form for 10 min at roomtemperature. The transfection mixture then was slowly added to the 100ml culture of CHO-S cells while gently swirling the flask. Similarly,37.5 μg RSLV-606 and 37.5 μg RSLV-611; and 37.5 μg RSLV-608 and 37.5 μgRSLV-613, were co-transfected into 60 ml of CHO-S cultures.

The cultures were incubated on an orbital shaker platform (120-135 rpm)at 37° C. in an 8% CO2 incubator. After 7 days of growth, cells wereharvested by centrifugation (1000 rpm for 10 min) and the conditionedmedium was recovered and filtered by passage through a 0.22 um membrane.

Each clarified culture medium (co-transfected RSLV-601/604,RSLV-602/603, RSLV-606/611, and RSLV-608/613) was purified by passingover a protein-A column (Bio-Rad BioScale Mini Protein A, Catalog#7324600) and washed with 10 column volumes of PBS buffer at pH 7.2. Thebound material was eluted with 5 column volumes of citrate buffer at pH3.6 with each fraction neutralized with TRIS at pH 11. The fractionscontaining protein were pooled and equilibrated into PBS by dialysisusing a 10 k MWCO dialysis unit.

Western Blot Analysis: Expression of the soluble interferon receptorswas assayed by standard Western Blot analysis. Based on estimatedprotein concentration, 4 μg of protein were loaded into each lane andthe samples electrophoresed on Novex 4-20% Tris Glycine gradient gelunder denaturing conditions+/−reducing agent (R, NR). Purified rhuIFNAR1 and rhu IFNAR2 samples (Sino Biological, Catalog #13222-H08H and#10359-H08H) were run as controls. Duplicate gels were prepared and eachgel contained a single lane of molecular weight standards. Followingelectrophoresis, proteins were blotted onto nitrocellulose and the blotssubsequently blocked by incubation in Odyssey Blocking Buffer for 1 hr.Blot 1 was then exposed sequentially to: 0.1 μg/ml of polyclonal goatanti human IFNAR1 antibody (R&D Systems, Catalog #AF245), and a 1:10,000dilution of Dylight 800 conjugated donkey anti goat IgG (Rockland,Catalog #605-745-002). Blot 2 was exposed sequentially to 1 μg/ml ofpolyclonal sheep anti human IFNAR2 (R&D Systems, Catalog #AF4015), and a1:50,000 dilution of Alexa Fluor 680-conjugated AffiniPure DonkeyAnti-Sheep IgG (Jackson, Catalog #713-625-147). The blots were imagedusing a Licor Odyssey.

Expression of the RSLV 601-604 and RSLV 602-603 soluble interferonreceptors was observed to run slightly higher than the predictedmolecular weight, possibly due to glycosylation of the proteins (datanot shown).

SDS-PAGE, Coomassie Blue: The soluble interferon receptors werepurified, electrophoresed, and visualized using Coomassie blue.Fractions generated during Protein A purifications of conditioned mediumharvested from RSLV 601-604, RSLV 602-603, and RSLV 608-613co-transfections in CHO-S cells were visualized by SDS-PAGE withCoomassie Blue staining. 15 ul of each sample was diluted with 5 ul of4× Protein Loading Buffer and heat denatured. Samples were applied toNovex 4-20% Tris Glycine gradient gels and stained with Simply Blue Safestain following electrophoresis, and imaged using a Licor Odyssey. Eachgel had a single lane of MW standards for reference.

Positive fractions identified were pooled and equilibrated into PBS bydialysis using a 10 k MWCO dialysis cassette. Protein concentrationestimates were determined by OD₂₈₀ values. 500 ng of each solubleinterferon receptor (RSLV 601-604, RSLV 602-603, and RSLV608-613)+/−reducing agent were applied to a Novex 4-20% Tris Glycinegradient gel and stained with Simply Blue Safe stain followingelectrophoresis, and imaged using Licor Odyssey.

A major band was detected in the starting material and in fractions 2-5of the purified RSLV 602-603 soluble interferon receptor (data notshown). The theoretical mass of the RSLV-602-603 soluble interferonreceptor is 130,754 Daltons and ran higher than predicted on SDS-PAGEpossibly due to glycosylation. Likewise, a major band was detected inthe starting material and in fractions 2-4 of the purified RSLV 601-604soluble interferon receptor (data not shown). The theoretical mass ofthe RSLV-601-604 soluble interferon receptor is 130,754 Daltons and ranhigher than predicted on SDS-PAGE possibly due to glycosylation. Similarresults were obtained for RSLV-608-613 as well as RSLV-606 and RSLV-611(data not shown). Reduced and non-reduced fractions of Protein Apurified RSLV 608-613 were subjected to SDS-PAGE analysis and theidentity and molecular weight of RSLV 608-613 expressed in CHOsupernatants was verified by western blot using anti-IFNAR antibodies(data not shown).

Example 3 Inhibition of IFNα Activity

The soluble interferon receptors were assessed for their ability toinhibit IFNα induced secreted alkaline phosphatase (SEAP) production inHEK-Blue α/β cells.

HEK-Blue IFN-α/β cells (Invivogen, Catalog #hkb-ifnab) produce andsecrete SEAP in response to stimulation by IFN-α or IFN-β. To assessinhibition of IFN-α activity, replicate titrations of inhibitors (RSLV601-604, RSLV 602-603, and anti-human IFNα control) were prepared in a96 well plate. A fixed concentration of IFNα (0.2 ng/ml finalconcentration) was added to the wells. HEK-Blue IFN-α/β cells were thenadded to the plate at 50,000 cells/well and plates were incubated 20-24hours at 37° C. in 5% CO₂. 20 μl of cell supernatant was then added to180 μl of QUANTI-Blue reagent in a 96 well tissue culture plate, andincubated for 1-3 hr at 37° C. SEAP activity was then detected bymeasuring absorbance at 620 nm.

The inhibition assays of IFNα activity were performed in triplicate. Asshown in FIG. 2, both RSLV 601-604 and RSLV 602-603 were able to inhibitIFNα induced SEAP production to a similar extent as the anti-IFNαcontrol molecule. RSLV 601-604 had an IC₅₀ value of 0.534 nM, RSLV602-603 had an IC₅₀ value of 0.461 nM, and the anti-IFNα control had anIC₅₀ value of 0.138 nM.

Example 4 Inhibition of IFN Activity

The soluble interferon receptors were assessed for their ability toinhibit IFNβ induced secreted alkaline phosphatase (SEAP) production inHEK-Blue α/β cells.

HEK-Blue IFN-α/β cells (Invivogen, Catalog #hkb-ifnab) produce andsecrete SEAP in response to stimulation by IFN-α or IFN-β. To assessinhibition of IFN-β activity, replicate titrations of inhibitors (RSLV601-604, RSLV 602-603, and anti-human IFNβ control) were prepared in a96 well plate. A fixed concentration of IFNβ (5 pg/ml finalconcentration) was added to the wells. HEK-Blue IFN-α/β cells were thenadded to the plate at 50,000 cells/well and plates were incubated 20-24hours at 37° C. in 5% CO₂. 20 μl of cell supernatant was then added to180 μl of QUANTI-Blue reagent in a 96 well tissue culture plate, andincubated for 1-3 hours at 37° C. SEAP activity was then detected bymeasuring absorbance at 620 nm.

Four replicates of the inhibition assays of IFNβ activity wereperformed. As shown in FIG. 3, both RSLV 601-604 and RSLV 602-603 wereable to inhibit IFNβ induced SEAP production to a greater extent thanthe anti-IFNβ control molecule. RSLV 601-604 had an IC₅₀ value of 0.020nM, RSLV 602-603 had an IC₅₀ value of 0.015 nM, and the anti-IFNβcontrol had an IC₅₀ value of 0.059 nM.

Example 5 Impact of Linker Length on the Inhibition of IFN-α Activity

The impact of linker length on the ability of the soluble interferonreceptors to inhibit IFN-α was assayed according to the methodsdescribed in Example 3. The inhibitors used in the assay were RSLV601-604, RSLV 606-611, RSLV 608-613, and anti-human IFNα control. RSLV601-604 has a (Gly₄Ser)₄ linker; RSLV 606-611 has a (Gly₄Ser)₂ linker,and RSLV 608-613 does not include a linker domain.

The inhibition assays of IFNα activity were performed twice. As shown inFIG. 4, all constructs were able to inhibit IFNα induced SEAPproduction, although the constructs without linkers or shortened linkersappear to be more potent. For example, RSLV 601-604 had an IC₅₀ value of1.051 nM, RSLV 606-611 had an IC₅₀ value of 0.835 nM, and RSLV 608-613had an IC₅₀ value of 0.375. Anti-human IFNα control had an IC₅₀ value of0.640 nM. When the constructs with different linkers were compared toeach other, it was observed that IFN-α binding affinity and potencyincreased as linker length decreased.

Example 6 Impact of Linker Length on the Inhibition of IFN-β Activity

The impact of linker length on the ability of the soluble interferonreceptors to inhibit IFN-β was assayed according to the methodsdescribed in Example 4. The inhibitors used in the assay were RSLV601-604, RSLV 606-611, RSLV 608-613, and anti-human IFNα control. RSLV601-604 has a (Gly₄Ser)₄ linker; RSLV 606-611 has a (Gly₄Ser)₂ linker,and RSLV 608-613 does not include a linker domain.

The inhibition assays of IFNβ activity were performed twice. As shown inFIG. 5, all constructs were able to inhibit IFNβ induced SEAP productionto a greater extent than the anti-IFNβ control molecule. RSLV 601-604had an IC₅₀ value of 0.011 nM, RSLV 606-611 had an IC₅₀ value of 0.015nM, and RSLV 608-613 had an IC₅₀ value of 0.010. Anti-human IFNβ controlhad an IC₅₀ value of 0.043 nM. When the constructs with differentlinkers were compared to each other, it was observed that linker lengthdid not have an impact on IFN-β binding affinity and potency.

Example 7 Impact of Soluble Interferon Receptor Constructs on theInhibition of SLE Sera Induced Interferon Gene Expression in PBMC

To assess whether interferon (IFN) gene expression could be inhibited bythe soluble interferon receptor constructs, interferon regulated geneswere induced in peripheral blood mononuclear cells (PBMC) obtained froma health volunteer using systemic lupus erythematosus (SLE) serum frompatients that were previously identified as IFN gene signature positive.For example, a three gene (HERC5, EPSTI, and CMPK2) surrogate(interferon signature metric (ISM)) for the interferon signature (IS)can be used as a biomarker to distinguish patients with SLE onserological characteristics (Kennedy et al., Lupus Science and Medicine,2015; 2:e000080. Doi10.1136/lupus-2014-000080, the entire contents ofwhich is incorporated herein by reference).

Thawing of PBMC

PBMC from a single healthy volunteer were thawed according to thefollowing protocol: RPMI complete media was made by adding 10%heat-inactivated FBS and 1% pen/strep. Nine ml of RPMI complete wasaliquoted into a 15-ml conical for each cryovial to be thawed. Eachcyrovial was partially submerged in a 37° C. water bath and moved backand forth to partially thaw the vial. The vial was then removed from thewater bath and the exterior was sprayed with 70% ethanol. The cryovialwas then uncapped and 1 ml of RPMI complete was added to the vialdropwise. The thawed PMBC were then transferred to the 9 ml aliquot ofRPMI complete. The PBMC cells in RPMI complete were then centrifuged at300×g for 7-10 minutes at room temperature. The supernantent was removedfrom the cells and the cells were suspended in RPMI complete to 2×10⁶cells/ml. the cells were then transferred to a 25 ml tissue cultureflask and allowed to rest overnight in 5% CO₂ at 37° C.

Stimulation of PBMC with SLE Patient Sera+/−RSLV Inhibitors

PBMC were stimulated with SLE patient sera from six patients (with orwithout the RSLV 608-613 inhibitor or with or without the RSLV601-604inhibitor) according to the following protocol: SLE patient sera waspre-incubated with the RSLV inhibitor by adding 15 μg RSLV 608-613 (27μl of RSLV-608-613 at 555 μg/ml) or 15 μg RSLV 601-604 (50 μl of RSLV601-604 at 306 μg/ml) to 200 μl SLE patient sera in duplicate wells of a24-well plate. For SLE patient sera without inhibitors, 27 μl of RPMIcomplete was added to 200 μl of SLE patient sera to duplicate wells of a24-well plate. The samples were mixed gently and incubated for 30minutes at 37° C. During the 30 minute incubation, PBMC were harvestedfrom the 25 ml tissue culture flasks an centrifuged to pellet. The PBMCwere counted and resuspended to 2×10⁷ cells/ml in RPMI complete. Theresuspended PBMC were added to wells containing the SLE serum andincubated for six hours in 5% CO₂ at 37° C.

Preparation of RNA from Stimulated PBMC Using RNeasy Plus Mini Kit

Following incubation of the PBMCs with or without the RSLV constructs,RNA from the stimulated PBMC was prepared using the RNeasy® Plus MiniKit from Qiagen® according to the manufacturer's protocol. PBMC wereharvested from each well of the 24-well plate into 1.5 ml Eppendorftubes and centrifuged for 2 minutes at 1000×g to pellet the cells. Thesupernatant was aspirated from the tubes and the pelleted cells wereretained. Any remaining PBMC in the wells of the 24-well plate werelysed with 350 ml buffer RLT plus. The cell lysates were then added tothe appropriate cell pellet and vortexed for 30 seconds. The homogenizedlysate was transferred to a gDNA Eliminator spin column and placed in a2 ml collection tube. The tubes were centrifuges for 30 seconds at≥8000×g (≥10,000 rpm). The flow-though was saved and the column wasdiscarded. 350 μl of 70% ethanol was added to the flow-through and mixedwell by pipetting. 700 μl of the sample, including any precipitate wasimmediately transferred to a RNeasy spin column and placed in a 2 mlcollection tube. The tubes were centrifuged for 15 seconds at >8,000×gand the flow-though was discarded. 700 μl of buffer RW1 was added to theRNeasy Mini spin column (in a 2 ml collection tube), the lids wereclosed, and the tubes were centrifuged for 15 seconds at ≥8,000×g. Theflow-through was discarded. 500 μl of buffer RPE was added to the RNeasyMini spin column (in a 2 ml collection tube), the lids were closed, andthe tubes were centrifuged for 15 seconds at ≥8,000×g. The flow-throughwas discarded. 500 μl of buffer RPE was added to the RNeasy Mini spincolumn (in a 2 ml collection tube), the lids were closed, and the tubeswere centrifuged for 2 minutes at ≥8,000×g. The flow-through wasdiscarded. The RNeasy spin column was then placed in a new 2 mlcollection tube and centrifuged at full speed for 1 minute to furtherdry the membrane. The RNeasy spin column was then placed in a new 1.5 mlcollection tube and 30 μl of RNase-free water was added directly to thespin column membrane. The lids were closed and the tubes were thencentrifuged for 1 minute at >8,000×g to elute the RNA.

cDNA Synthesis

The RNA derived from the PBMCs treated with or without the RSLVconstructs was converted into cDNA as follows: First-strand cDNA wasgenerated from the isolated RNA (as described above) using theSuperScript VILO cDNA synthesis kit (Life Technologies). cDNA wassynthesized from 10 ng of RNA in a 20μl reaction. A control withoutreverse transcriptase was also performed for each sample. RNA wasquantitated based on absorbance at 260 nm on a Nanodrop 2000spectrophotometer (ThermoFisher) using a conversion factor of 1A₂₆₀=40μg/ml. 10 ng RNA was used as input for cDNA synthesis.

The cDNA reaction mixture was prepare for all reactions to be run. Themix for a single reaction consists of: 4μl 5×VILO Reaction Mix, 2 μl 10×SuperScript Enzyme Mix, and 10 μl molecular grade water. Enough Reactionmix lacking the 10× Enzyme mix was prepared for use with one RNA samplefrom each sample (patient sample with or without the RSLV inhibitor, IFNpositive RNA control, and IFN negative RNA control). The cDNA reactionplate was placed on ice, and for each reaction 16 μl of the appropriatereaction mix was added to the desired well of a 96 well QPCR plate. 4 μlof RNA was added to each reaction well and mixed by pipetting up anddown several times. The wells were capped and the plate was transferredto a thermal cycler and incubated at 25° C. for 10 minutes, followed by42° C. for 1 hour, followed by 80° C. for five minutes. 80 μl of RNasefree water was added to each cDNA reaction and mixed by pipetting up anddown several times.

QPCR Measurement of Interferon Signature

QPCR (Taqman) was used to measure the levels of threeinterferon-inducible genes (HERC5, EPSTI1, and CMPK2) and threereference genes (HPRT1, GUSB, and TFRC) present in the cDNAs preparedabove. 1 μl of 1 mM ROX reference dye (provided with Brilliant MultiplexMasters Mix) was added to to 500 μl water to produce a 204 stocksolution. QPCR reaction mix was prepared for all reactions.

The QPCR reaction mix was divided into seven aliquots with enoughreaction mix for each of the six primer/probe sets (sequences shownbelow) plus an additional aliquot of mix for a set of no RT controls. Asingle primer/probe set was added to each aliquot. The stockconcentration of the primer probe sets was 40×. Therefore, 0.625 μl ofprimer/probe was used for each 25 μl reaction. A primer and probe setwas prepared for each of the six genes, and a single primer/probe setwas prepared for the no RT control wells. 20 μl of each reaction mixturewas transferred to the wells of a QPCR 96 well plate. 5 μl of each cDNAsample was loaded into the QPCR wells containing each primer/probe mix,and mixed thoroughly by pipetting up and down. All wells were cappedwith QPCR strip caps, and the plate was spun briefly at 1000 rpm. Theplate was loaded into an Mx3005p QPCR instrument and run according tothe following cycling conditions: 95° C. for 10 minutes, followed by 40cycles of: 95° C. for 15 seconds and 60° C. for 1 minute. The followingdetection settings were used: data was collected using FAM, and ROXfilter sets at the end of each 60° C. step (filter gain settings: ROX×1,FAM×8).

TFRC Probe /56-FAM/CCA TTG TCA/ZEN/TAT ACC CGG TTC AGC CT/3IABkFQ/TFRC Primer 1 ATC TAC AGC AAG TTT CAT CTC CA TFRC Primer 2TCA AGC TAG ATC AGC ATT CTC TAA C HPRT1 Probe/56-FAM/TCC ATT CCT/ZEN/ATG ACT GTA GAT TTT ATC AGA CTG AAG A/3IABkFQ/HPRT1 Primer 1 CCA ATT ACT TTT ATG TCC CCT GTT HPRT1 Primer 2CAT CAA AGC ACT GAA TAG AAA TAG TGA GUSB Probe/56-FAM/TGC AGG GTT/ZEN/TCA CCA GGA TCC AC/3IABkFQ/ GUSB Primer 1GTT TTT GAT CCA GAC CCA GAT G GUSB Primer 2 GCC CAT TAT TCA GAG CGA GTACMPK2 Probe /56-FAM/ATG CCA CGG/ZEN/GTA AAA CCA CGG T/3IABkFQ/CMPK2 Primer 1 AGG ACA GCC TTA AGT GAA TCT G CMPK2 Primer 2GCC CAA AAC AGA TCC AGA AAG HERC5 Probe/56-FAM/ATA CCC AAC/ZEN/AAG CTC AGC CAC CA/3IABkFQ/ HERC5 Primer 1CCC AAA TCA GAA ACA TAG GCA AG HERC5 Primer 2TCA ACA CAG AAT GAG CTA AGA CC EPSTI1 Probe/56-FAM/AGA GCC AAA/ZEN/ATC CAC CAG ACT GAA CA/3IABkFQ/ EPSTI1 Primer 1TCC AAC AGC CTC CAG ATT G EPSTI1 Primer 2 GTG AAT TAC TGG AAC TGA AAC GG

Data Analysis

The QPCR data was then analyzed. Cycle threshold (Ct) values for eachQPCR reaction were obtained using MxPro version 4.10 software. Thresholdfluorescence values were set automatically by the software withamplification-based threshold, adaptive baseline, and moving averageoptions checked. For each sample the log₂ scaled relative expression ofIFN-regulated genes was calculated as the mean Ct of the 3 IFN-regulatedgenes (CMPK2, HERC5, and EPSTI1) minus the mean Ct of the 3 referencegenes (GUSB, HPRT1, and TFRC). This quantity was multiplied by −1 togive the correct directionality to the value.

Summary

As shown in FIG. 6, RSLV-601-604 inhibited SLE sera induced interferongene expression in PBMC from each of the six SLE patients. Similarly,RSLV 608-613 also inhibited SLE patient sera induced interferon genestimulation (FIG. 7) These data demonstrate that the soluble interferonreceptor constructs are effective at inhibiting the cytokines in SLEserum which are responsible for inducing interferon gene expression.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

TABLE 1 SEQUENCE LISTING SEQ ID NO: DESCRIPTION SEQUENCE 1 RSLV-601MDWTWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD Leader-IFNAR1ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFSSLKLNVYE (aa 28-436)-EIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV (Gly4Ser)4-IHISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHK IgG1 FcIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPE (aa 216-NIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWK 447) withQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIK mutationsFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLI C220S, P238S,YEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDE P331S, T350V,KLNKSSVFSDAVCEKTKPGNTSKGGGGSGGGGSGGGGSGGGGSEP T366L, K392L,KSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV T394WVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 2 RSLV-602MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNFRSIL Leader-IFNAR2SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD (aa 27-243)-EWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMSFEPPEFEIV (Gly4Ser)4-GFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG IgG1 Fc (aaNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPP 216-447) withGQESESAESAKGGGGSGGGGSGGGGSGGGGSEPKSSDKTHTCPPC mutationsPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK C220S, P238S,FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY P331S, T350V,KCKVSNKALPASIEKTISKAKGQPREPQVYVLPPSRDELTKNQVS T366L, K392L,LLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSK T394WLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 3 RSLV-603MDWTWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD Leader-IFNAR1ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFSSLKLNVYE (aa 28-436)-EIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV (Gly4Ser)4-IHISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHK IgG1 Fc (aaIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPE 216-447) withNIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWK mutationsQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIK C220S, P238S,FDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLI P331S, T350V,YEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDE L351Y, F405A,KLNKSSVFSDAVCEKTKPGNTSKGGGGSGGGGSGGGGSGGGGSEP Y407VKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 4 RSLV-604MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNFRSIL Leader-IFNAR2SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD (aa 27-243)-EWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMSFEPPEFEIV (Gly4Ser)4-GFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG IgG1 Fc (aaNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPP 216-447) withGQESESAESAKGGGGSGGGGSGGGGSGGGGSEPKSSDKTHTCPPC mutationsPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK C220S, P238S,FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY P331S, and ZW1KCKVSNKALPASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVS Chain ALTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSK mutationsLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK T350V, L351Y, F405A, Y407V 5IFNAR1 MMVVLLGATTLVLVAVAPWVLSAAAGGKNLKSPQKVEVDIIDDNF ACCESSIONILRWNRSDESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFS NP_000620SLKLNVYEEIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKIWLIVGICIALFALPFVIYAAKVFLRCINYVFFPSLKPSSSIDEYFSEQPLKNLLLSTSEEQIEKCFIIENISTIATVEETNQTDEDHKKYSSQTSQDSGNYSN EDESESKTSEELQQDFV 6 IFNAR2MLLSQNAFIFRSLNLVLMVYISLVFGISYDSPDYTDESCTFKISL ACCESSIONRNFRSILSWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTR NP_997468SFCDLTDEWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKIGGIITVFLIALVLTSTIVTLKWIGYICLRNSLPKVLNFHNFLAWPFPNLPPLEAMDMVEVIYINRKKKVWDYNYDDESDSDTEAAPRTSGGGYTMHGLTVRPLGQASATSTESQLIDPESEEEPDLPEVDVELPTMPKDSPQQLELLSGPCERRKSPLQDPFPEEDYSSTEGSGGRITFNVDLNSVFLRVLDDEDSDDLEAPLMLSSHLEEMVDPEDPDNVQSNHLLASGEGTQPTFPSPSSEGLWSEDAP SDQSDTSESDVDLGDGYIMR 7IFNAR1 MMVVLLGATTLVLVAVAPWVLSAAAGGKNLKSPQKVEVDIIDDNF ExtracellularILRWNRSDESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFS domain (withSLKLNVYEEIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHL signalEAEDKAIVIHISPGTKDSVMWALDGLSFTYSLLIWKNSSGVEERI sequence)ENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSK 8 IFNAR2MLLSQNAFIFRSLNLVLMVYISLVFGISYDSPDYTDESCTFKISL ExtracellularRNFRSILSWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTR domainSFCDLTDEWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMSFE (with signalPPEFEIVGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKK sequence)HKPEIKGNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPL KCTLLPPGQESESAESAK 9IgG1 Fc (aa EPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVT 216-447) withCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV mutationsLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYV C220S, P238S,YPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP P331S, T350V,PVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS L351Y, F405A, LSLSPGKY407V 10 IgG1 Fc EPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVT (aa 216-CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV 447) withLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYV mutationsLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWP C220S, P238S,PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS P331S, T350V, LSLSPGKT366L, K392L, T394W 11 IFNAR1 (aa 28-KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN 436)WIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPG NTSK 12 IFNAR2 (aa 27-ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT 243)IMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAK 13 Leader MDWTWRILFLVAAATGTHASequence 14 VK3LP leader METPAQLLFLLLLWLPDTTG sequence 15 (Gly4Ser)4GGGGSGGGGSGGGGSGGGGS 16 (Gly4Ser)3 GGGGSGGGGSGGGGS 17 (Gly4Ser)5GGGGSGGGGSGGGGSGGGGSGGGGS 18 NLG linker VDGASSPVNVSSPSVQDI 19 linkerLEA(EAAAK)₄ALEA(EAAAK)₄ALE 20 IgG1 Fc (SCCEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT hinge)CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 21 IgG1 Fc domainEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVT (SSS hinge)CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 22 IgG1 Fc domainEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVT with P238SCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV (SCC hinge)LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 23 IgG1 Fc domainEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT with P331SCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 24 IgG1 Fc domainEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT with SSS,CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV P238S, andLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYT P331SLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 25 IgG1 Fc domainEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVT with SCC,CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV P238S, andLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYT P331SLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 26 Human IgG1 FcEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT domain (wild-CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV type)LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 27 linker GGSG 28linker GSAT 29 O-linked CXXGGT/S-C glycosylation consensus site 30O-linked NST-E/D-A glycosylation consensus site 31 O-linked NITQSglycosylation consensus site 32 O-linked QSTQS glycosylationconsensus site 33 O-linked D/E-FT-R/K-V glycosylation consensus site 34O-linked C-E/D-SN glycosylation consensus site 35 O-linked GGSC-K/Rglycosylation consensus site 36 IFNAR1KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN (withoutWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEV signalDSFTPFRKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS sequence)FTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKIWLIVGICIALFALPFVIYAAKVFLRCINYVFFPSLKPSSSIDEYFSEQPLKNLLLSTSEEQIEKCFIIENISTIATVEETNQTDEDHKKYSSQTSQDSGNYSNEDESESKTSEELQQDFV 37 IFNAR2ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT (withoutIMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGN signalTTLFSCSHNFWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE sequence)LQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKIGGIITVFLIALVLTSTIVTLKWIGYICLRNSLPKVLNFHNFLAWPFPNLPPLEAMDMVEVIYINRKKKVWDYNYDDESDSDTEAAPRTSGGGYTMHGLTVRPLGQASATSTESQLIDPESEEEPDLPEVDVELPTMPKDSPQQLELLSGPCERRKSPLQDPFPEEDYSSTEGSGGRITFNVDLNSVFLRVLDDEDSDDLEAPLMLSSHLEEMVDPEDPDNVQSNHLLASGEGTQPTFPSPSSEGLWSEDAPSDQSDTSESDVDLGDGYIMR 38 (Gly4Ser)2 GGGGSGGGGS 39(Gly4Ser)1 GGGGS 40 RSLV-601KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN Without aWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEV leaderDSFTPFRKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS sequenceFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKGGGGSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 41 RSLV-602ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT Without aIMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGN leaderTTLFSCSHNFWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE sequenceLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKGGGGSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 42RSLV-603 KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN Without aWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEV leaderDSFTPFRKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS sequenceFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKGGGGSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 43 RSLV-604ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT without aIMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGN leaderTTLFSCSHNFWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE sequenceLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKGGGGSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 44Leader-IFNAR2 MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNFRSIL (aa 27-243)-SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD (Gly4Ser)2-EWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMSFEPPEFEIV IgG1 Fc (aaGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG 216-447) withNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPP mutationsGQESESAESAKGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGSS C220S, P238S,VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV P331S, T350V,HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP T366L, K392L,ASIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYP T394WSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 45 IFNAR2 (aa 27-ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT 243)-IMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGN (Gly4Ser)2-TTLFSCSHNFWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE IgG1 Fc (aaLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNY 216-447) withCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKGGGGSGGG mutationsGSEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPE C220S, P238S,VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV P331S, T350V,SVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQV T366L, K392L,YVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLT T394W, withoutWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ a leader KSLSLSPGKsequence 46 Leader-IFNAR2 MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNFRSIL(aa 27-243)- SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD IgG1 Fc (aaEWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMSFEPPEFEIV 216-447) withGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG mutationsNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPP C220S, P238S,GQESESAESAKEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKD P331S, T350V,TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE T366L, K392L,QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKA T394WKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 47IFNAR2 (aa 27- ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT243)-IgG1 Fc IMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGN (aa 216-TTLFSCSHNFWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE 447) withLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNY mutationsCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKEPKSSDKT C220S, P238S,HTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSH P331S, T350V,EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW T366L, K392L,LNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVLPPSRDEL T394W, withoutTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGS a leaderFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence 48 Leader-IFNAR1MDWTWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD (aa 28-436)-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFSSLKLNVYE (Gly4Ser)2-EIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV IgG1 Fc (aaIHISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHK 216-447) withIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPE mutationsNIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWK C220S, P238S,QIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIK P331S, T350V,FDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLI L351Y, F405A,YEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDE Y407VKLNKSSVFSDAVCEKTKPGNTSKGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 49 IFNAR1 (aa 28-KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN 436)-WIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEV (Gly4Ser)2-DSFTPFRKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS IgG1 Fc (aaFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALL 216-447) withTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTY mutationsANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNV C220S, P238S,FQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSL P331S, T350V,SDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEK L351Y, F405A,KTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPG Y407V, withoutNTSKGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGSSVFLFPPK a leaderPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP sequenceREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 50Leader-IFNAR1 MDWTWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD (aa 28-436)-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFSSLKLNVYE IgG1 Fc (aaEIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV 216-447) withIHISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHK mutationsIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPE C220S, P238S,NIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWK P331S, T350V,QIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIK L351Y, F405A,FDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLI Y407VYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 51 IFNAR1 (aa 28-KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN 436)-IgG1 FcWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEV (aa 216-DSFTPFRKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS 447) withFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALL mutationsTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTY C220S, P238S,ANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNV P331S, T350V,FQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSL L351Y, F405A,SDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEK Y407V, withoutKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPG a leaderNTSKEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRT sequencePEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 52 RSLV-606MDWTWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD Leader-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFSSLKLNVYE IFNAR1 (aa 28-EIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV 436)-IHISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHK (Gly4Ser)2- FcIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPE (aa 216-NIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWK 447) withQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIK mutationsFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLI C220S, P238S,YEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDE P331S, T350V,KLNKSSVFSDAVCEKTKPGNTSKGGGGSGGGGSEPKSSDKTHTCP T366L, K392L,PCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE T394WVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 53 RSLV-606KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN Without aWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEV leaderDSFTPFRKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS sequenceFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 54RSLV-611 MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNFRSIL Leader-IFNAR2SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD (aa 27-243)-EWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMSFEPPEFEIV (Gly4Ser)2-FcGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG (aa 216-447)NMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPP with mutationsGQESESAESAKGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGSS C220S, P238S,VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV P331S, T350V,HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP L351Y, F405A,ASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYP Y407VSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 55 RSLV-611ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT Without aIMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGN leaderTTLFSCSHNFWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE sequenceLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 56 RSLV-608MDWTWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD Leader-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFSSLKLNVYE IFNAR1 (aa 28-EIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV 436)-FcIHISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHK (aa 216-IYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPE 447) withNIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWK mutationsQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIK C220S, P238S,FDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLI P331S, T350V,YEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDE T366L, K392L,KLNKSSVFSDAVCEKTKPGNTSKEPKSSDKTHTCPPCPAPELLGG T394WSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 57 RSLV-608KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN Without aWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEV leaderDSFTPFRKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS sequenceFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 58 RSLV-613MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNERSIL Leader-IFNAR2SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD (aa 27-243)- FcEWRSTHEAYVTVLEGFSGNTTLFSCSHNEWLAIDMSFEPPEFEIV (aa 216-447)GFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG with mutationsNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPP C220S, P238S,GQESESAESAKEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKD P331S, T350V,TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE L351Y, F405A,QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKA Y407VKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVESCSVMH EALHNHYTQKSLSLSPGK 59RSLV-613 ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT Without aIMSKPEDLKVVKNCANTTRSECDLTDEWRSTHEAYVTVLEGFSGN leaderTTLFSCSHNEWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE sequenceLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 60 Leader-MDWTWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD IFNAR1 (aa 28-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNESSLKLNVYE 436)-EIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV (Gly4Ser)4-IHISPGTKDSVMWALDGLSETYSLVIWKNSSGVEERIENIYSRHK IgG1 Fc domainIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPE with T366YNIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWK mutationQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAELLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVESDAVCEKTKPGNTSKGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLS LSPGK 61 IFNAR1 (aa 28-KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN 436)-WIKLSGCQNITSTKCNESSLKLNVYEEIKLRIRAEKENTSSWYEV (Gly4Ser)4-DSFTPERKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS IgG1 Fc domainFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALL with T366YTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTY mutationANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNV without aFQKGIYLLRVQASDGNNTSFWSEEIKEDTEIQAFLLPPVFNIRSL leaderSDSFHIYIGAPKQSGNTPVIQDYPLIYETIFWENTSNAERKIIEK sequenceKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 62 Leader-IFNAR2MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNERSIL (aa 27-243)-SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD (Gly4Ser)4-EWRSTHEAYVTVLEGFSGPITTLFSCSHNEWLAIDMSFEPPEFEIV IgG1 Fc domainGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG with T366YNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPP mutationGQESESAESAKGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 63 IFNAR2 (aa 27-ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT 243)-IMSKPEDLKVVKNCANTTRSECDLTDEWRSTHEAYVTVLEGFSGN (Gly4Ser)4-TTLFSCSHNEWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE IgG1 Fc domainLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNY with T366YCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKGGGGSGGG mutationGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPK without aDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE leaderEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK sequenceAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVM HEALHNHYTQKSLSLSPGK 64Leader-IFNAR1 MDWTWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD (aa 28-436)-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNESSLKLNVYE (Gly4Ser)4-EIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV IgG1 Fc domainIHISPGTKDSVMWALDGLSETYSLVIWKNSSGVEERIENIYSRHK with Y407TIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPE mutationNIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAELLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVESDAVCEKTKPGNTSKGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELTSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLS LSPGK 65 IFNAR1 (aa 28-KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN 436)-WIKLSGCQNITSTKCNESSLKLNVYEEIKLRIRAEKENTSSWYEV (Gly4Ser)4-DSFTPERKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS IgG1 Fc domainFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALL with Y407TTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTY mutationANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNV without aFQKGIYLLRVQASDGNNTSFWSEEIKEDTEIQAFLLPPVFNIRSL leaderSDSFHIYIGAPKQSGNTPVIQDYPLIYETIFWENTSNAERKIIEK sequenceKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 66 Leader-IFNAR2MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNERSIL (aa 27-243)-SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD (Gly4Ser)4-EWRSTHEAYVTVLEGFSGNTTLFSCSHNEWLAIDMSFEPPEFEIV IgG1 Fc domainGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG with Y407TNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPP mutationGQESESAESAKGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 67 IFNAR2 (aa 27-ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT 243)-IMSKPEDLKVVKNCANTTRSECDLTDEWRSTHEAYVTVLEGFSGN (Gly4Ser)4-TTLFSCSHNEWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE IgG1 Fc domainLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNY with Y407TCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKGGGGSGGG mutationGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPK without aDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE leaderEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK sequenceAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVESCSVM HEALHNHYTQKSLSLSPGK 68Leader- MDWTWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD IFNAR1 (aa 28-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNESSLKLNVYE 436)-EIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV (Gly4Ser)2-IHISPGTKDSVMWALDGLSETYSLVIWKNSSGVEERIENIYSRHK IgG1 Fc domainIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPE with T366YNIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWK mutationQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAELLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVESDAVCEKTKPGNTSKGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 69 IFNAR1 (aa 28-KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN 436)-WIKLSGCQNITSTKCNESSLKLNVYEEIKLRIRAEKENTSSWYEV (Gly4Ser)2-DSFTPERKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS IgG1 Fc domainFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALL with T366YTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTY mutationANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNV without aFQKGIYLLRVQASDGNNTSFWSEEIKEDTEIQAFLLPPVFNIRSL leaderSDSFHIYIGAPKQSGNTPVIQDYPLIYETIFWENTSNAERKIIEK sequenceKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCS VMHEALHNHYTQKSLSLSPGK 70Leader-IFNAR2 MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNERSIL (aa 27-243)-SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD (Gly4Ser)2-EWRSTHEAYVTVLEGFSGNTTLFSCSHNEWLAIDMSFEPPEFEIV IgG1 Fc domainGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG with T366YNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPP mutationGQESESAESAKGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 71 IFNAR2 (aa 27-ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT 243)-IMSKPEDLKVVKNCANTTRSECDLTDEWRSTHEAYVTVLEGFSGN (Gly4Ser)2-TTLFSCSHNEWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE IgG1 Fc domainLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNY with T366YCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKGGGGSGGG mutationGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE without aVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV leaderSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV sequenceYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQ KSLSLSPGK 72 Leader-MDWIWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD IFNAR1 (aa 28-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNESSLKLNVYE 436)-EIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV (Gly4Ser)2-IHISPGTKDSVMWALDGLSETYSLVIWKNSSGVEERIENIYSRHK IgG1 Fc domainIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPE with Y407TNIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWK mutationQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAELLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVESDAVCEKTKPGNTSKGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 73 IFNAR1 (aa 28-KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN 436)-WIKLSGCQNITSTKCNESSLKLNVYEEIKLRIRAEKENTSSWYEV (Gly4Ser)2-DSFTPERKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS IgG1 Fc domainFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALL with Y407TTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTY mutationANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNV without aFQKGIYLLRVQASDGNNTSFWSEEIKEDTEIQAFLLPPVFNIRSL leaderSDSFHIYIGAPKQSGNTPVIQDYPLIYETIFWENTSNAERKIIEK sequenceKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVESCS VMHEALHNHYTQKSLSLSPGK 74Leader-IFNAR2 MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNERSIL (aa 27-243)-SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD (Gly4Ser)2-EWRSTHEAYVTVLEGFSGNTTLFSCSHNEWLAIDMSFEPPEFEIV IgG1 Fc domainGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG with Y407TNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPP mutationGQESESAESAKGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 75 IFNAR2 (aa 27-ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT 243)-IMSKPEDLKVVKNCANTTRSECDLTDEWRSTHEAYVTVLEGFSGN (Gly4Ser)2-TTLFSCSHNEWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE IgG1 Fc domainLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNY with Y407TCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKGGGGSGGG mutationGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE without aVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV leaderSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV sequenceYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVESCSVMHEALHNHYTQ KSLSLSPGK 76 Leader-MDWIWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD IFNAR1 (aa 28-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNESSLKLNVYE 436)-IgG1 FcEIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV domain withIHISPGTKDSVMWALDGLSETYSLVIWKNSSGVEERIENIYSRHK T366Y mutationIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAELLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVESDAVCEKTKPGNTSKEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 77 IFNAR1 (aa 28-KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN 436)-IgG1 FcWIKLSGCQNITSTKCNESSLKLNVYEEIKLRIRAEKENTSSWYEV domain withDSFTPERKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS T366Y mutationFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALL without aTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTY leaderANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNV sequenceFQKGIYLLRVQASDGNNTSFWSEEIKEDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYETIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHY TQKSLSLSPGK 78Leader-IFNAR2 MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNFRSIL (aa 27-243)-SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD IgG1 Fc domainEWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMSFEPPEFEIV with T366YGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG mutationNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 79IFNAR2 (aa 27- ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT243)-IgG1 Fc IMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGN domain withTTLFSCSHNFWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE T366Y mutationLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNY without aCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKEPKSCDKT leaderHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH sequenceEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 80 Leader-MDWIWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD IFNAR1 (aa 28-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFSSLKLNVYE 436)-IgG1 FcEIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV domain withIHISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHK Y407T mutationIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 81 IFNAR1 (aa 28-KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN 436)-IgG1 FcWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEV domain withDSFTPFRKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS Y407T mutationFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALL without aTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTY leaderANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNV sequenceFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK Leader-IFNAR2MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNERSIL (aa 27-243)-SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD IgG1 Fc domainEWRSTHEAYVTVLEGFSGNTTLFSCSHNEWLAIDMSFEPPEFEIV with Y407TGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG mutationNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVESCSVMH EALHNHYTQKSLSLSPGK 82IFNAR2 (aa 27- ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT243)-IgG1 Fc IMSKPEDLKVVKNCANTTRSECDLTDEWRSTHEAYVTVLEGFSGN domain withTTLFSCSHNEWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE Y407T mutationLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNY without aCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKEPKSCDKT leaderHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH sequenceEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 83 Leader-MDWTWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD IFNAR1 (aa 28-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNESSLKLNVYE 436)-EIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV (Gly4Ser)4-IHISPGTKDSVMWALDGLSETYSLVIWKNSSGVEERIENIYSRHK IgG1 Fc domainIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPE with T366WNIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWK mutationQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAELLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVESDAVCEKTKPGNTSKGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLS LSPGK 84 IFNAR1 (aa 28-KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN 436)-WIKLSGCQNITSTKCNESSLKLNVYEEIKLRIRAEKENTSSWYEV (Gly4Ser)4-DSFTPERKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS IgG1 Fc domainFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALL with T366WTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTY mutationANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNV without aFQKGIYLLRVQASDGNNTSFWSEEIKEDTEIQAFLLPPVFNIRSL leaderSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEK sequenceKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK Leader-IFNAR2MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNERSIL (aa 27-243)-SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD (Gly4Ser)4-EWRSTHEAYVTVLEGFSGNTTLFSCSHNEWLAIDMSFEPPEFEIV IgG1 Fc domainGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG with T366WNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPP mutationGQESESAESAKGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 85 IFNAR2 (aa 27-ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT 243)-IMSKPEDLKVVKNCANTTRSECDLTDEWRSTHEAYVTVLEGFSGN (Gly4Ser)4-TTLFSCSHNEWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE IgG1 Fc domainLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNY with T366WCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKGGGGSGGG mutationGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPK without aDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE leaderEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK sequenceAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVM HEALHNHYTQKSLSLSPGK 86Leader-IFNAR1 MDWTWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD (aa 28-436)-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNESSLKLNVYE (Gly4Ser)4-EIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV IgG1 Fc domainIHISPGTKDSVMWALDGLSETYSLVIWKNSSGVEERIENIYSRHK with T366S,IYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPE L368A, andNIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWK Y407VQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIK mutationsFDTEIQAELLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVESDAVCEKTKPGNTSKGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLS LSPGK 87 IFNAR1 (aa 28-KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN 436)-WIKLSGCQNITSTKCNESSLKLNVYEEIKLRIRAEKENTSSWYEV (Gly4Ser)4-DSFTPERKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS IgG1 Fc domainFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALL with T366S,TSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTY L368A, andANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNV Y407VFQKGIYLLRVQASDGNNTSFWSEEIKEDTEIQAFLLPPVFNIRSL mutationsSDSFHIYIGAPKQSGNTPVIQDYPLIYETIFWENTSNAERKIIEK without aKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPG leaderNTSKGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLG sequenceGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 88 Leader-IFNAR2MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNERSIL (aa 27-243)-SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD (Gly4Ser)4-EWRSTHEAYVTVLEGFSGNTTLFSCSHNEWLAIDMSFEPPEFEIV IgG1 Fc domainGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG with T366S,NMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPP L368A, andGQESESAESAKGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPC Y407VPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK mutationsFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 89 IFNAR2 (aa 27-ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT 243)-IMSKPEDLKVVKNCANTTRSECDLTDEWRSTHEAYVTVLEGFSGN (Gly4Ser)4-TTLFSCSHNEWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE IgG1 Fc domainLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNY with T366S,CVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKGGGGSGGG L368A, andGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPK Y407VDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE mutationsEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK without aAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWES leaderNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVESCSVM sequenceHEALHNHYTQKSLSLSPGK 90 Leader-MDWTWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD IFNAR1 (aa 28-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNESSLKLNVYE 436)-EIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV (Gly4Ser)2-IHISPGTKDSVMWALDGLSETYSLVIWKNSSGVEERIENIYSRHK IgG1 Fc domainIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPE with T366WNIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWK mutationQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAELLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVESDAVCEKTKPGNTSKGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 91 IFNAR1 (aa 28-KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN 436)-WIKLSGCQNITSTKCNESSLKLNVYEEIKLRIRAEKENTSSWYEV (Gly4Ser)2-DSFTPERKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS IgG1 Fc domainFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALL with T366WTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTY mutationANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNV without aFQKGIYLLRVQASDGNNTSFWSEEIKEDTEIQAFLLPPVFNIRSL leaderSDSFHIYIGAPKQSGNTPVIQDYPLIYETIFWENTSNAERKIIEK sequenceKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCS VMHEALHNHYTQKSLSLSPGK 92Leader-IFNAR2 MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNERSIL (aa 27-243)-SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD (Gly4Ser)2-EWRSTHEAYVTVLEGFSGNTTLFSCSHNEWLAIDMSFEPPEFEIV IgG1 Fc domainGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG with T366WNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPP mutationGQESESAESAKGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 93 IFNAR2 (aa 27-ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT 243)-IMSKPEDLKVVKNCANTTRSECDLTDEWRSTHEAYVTVLEGFSGN (Gly4Ser)2-TTLFSCSHNEWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE IgG1 Fc domainLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNY with T366WCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKGGGGSGGG mutationGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE without aVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV leaderSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV sequenceYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQ KSLSLSPGK 94 Leader-MDWIWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD IFNAR1 (aa 28-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNESSLKLNVYE 436)-EIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV (Gly4Ser)2-IHISPGTKDSVMWALDGLSETYSLVIWKNSSGVEERIENIYSRHK IgG1 Fc domainIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPE with T366S,NIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWK L368A, andQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIK Y407VFDTEIQAELLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLI mutationsYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVESDAVCEKTKPGNTSKGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 95 IFNAR1 (aa 28-KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN 436)-WIKLSGCQNITSTKCNESSLKLNVYEEIKLRIRAEKENTSSWYEV (Gly4Ser)2-DSFTPERKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS IgG1 Fc domainFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALL with T366S,TSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTY L368A, andANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNV Y407VFQKGIYLLRVQASDGNNTSFWSEEIKEDTEIQAFLLPPVFNIRSL mutationsSDSFHIYIGAPKQSGNTPVIQDYPLIYETIFWENTSNAERKIIEK without aKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPG leaderNTSKGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLEPPK sequencePKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVESCS VMHEALHNHYTQKSLSLSPGK 96Leader-IFNAR2 MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNERSIL (aa 27-243)-SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD (Gly4Ser)2-EWRSTHEAYVTVLEGFSGNTTLFSCSHNEWLAIDMSFEPPEFEIV IgG1 Fc domainGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG with T366S,NMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPP L368A, andGQESESAESAKGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPS Y407VVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV mutationsHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 97 IFNAR2 (aa 27-ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT 243)-IMSKPEDLKVVKNCANTTRSECDLTDEWRSTHEAYVTVLEGFSGN (Gly4Ser)2-TTLFSCSHNEWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE IgG1 Fc domainLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNY with T366S,CVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKGGGGSGGG L368A, andGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE Y407VVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV mutationsSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV without aYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT leaderTPPVLDSDGSFFLVSKLTVDKSRWQQGNVESCSVMHEALHNHYTQ sequence KSLSLSPGK 98Leader- MDWTWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD IFNAR1 (aa 28-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNESSLKLNVYE 436)-IgG1 FcEIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV domain withIHISPGTKDSVMWALDGLSETYSLVIWKNSSGVEERIENIYSRHK T366W mutationIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAELLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVESDAVCEKTKPGNTSKEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 99 IFNAR1 (aa 28-KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN 436)-IgG1 FcWIKLSGCQNITSTKCNESSLKLNVYEEIKLRIRAEKENTSSWYEV domain withDSFTPERKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS T366W mutationFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALL without aTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTY leaderANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNV sequenceFQKGIYLLRVQASDGNNTSFWSEEIKEDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYETIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHY TQKSLSLSPGK 100Leader-IFNAR2 MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNFRSIL (aa 27-243)-SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD IgG1 Fc domainEWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMSFEPPEFEIV with T366WGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG mutationNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDILMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 101IFNAR2 (aa 27- ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT243)-IgG1 Fc IMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGN domain withTTLFSCSHNFWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE T366W mutationLQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNY without aCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKEPKSCDKT leaderHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH sequenceEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 102 Leader-MDWIWRILFLVAAATGTHAKNLKSPQKVEVDIIDDNFILRWNRSD IFNAR1 (aa 28-ESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFSSLKLNVYE 436)-IgG1 FcEIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIV domain withIHISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHK T366S, L368A,IYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPE and Y407VNIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWK mutationsQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 103 IFNAR1 (aa 28-KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDN 436)-IgG1 FcWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEV domain withDSFTPFRKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLS T366S, L368A,FTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALL and Y407VTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTY mutationsANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNV without aFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSL leaderSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEK sequenceKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 104Leader-IFNAR2 MDWTWRILFLVAAATGTHAISYDSPDYTDESCTFKISLRNERSIL (aa 27-243)-SWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTD IgG1 Fc domainEWRSTHEAYVTVLEGFSGNTTLFSCSHNEWLAIDMSFEPPEFEIV with T366S,GFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKG L368A, andNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPP Y407VGQESESAESAKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD mutationsILMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVESCSVMH EALHNHYTQKSLSLSPGK 105IFNAR2 (aa 27- ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYT243)-IgG1 Fc IMSKPEDLKVVKNCANTTRSECDLTDEWRSTHEAYVTVLEGFSGN domain withTTLFSCSHNEWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEE T366S, L368A,LQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNY and Y407VCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAKEPKSCDKT mutationsHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH without aEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW leaderLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL sequenceTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 106 IgG1 Fc domainEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT with T366YCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV mutationLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT (knob hole 1)LPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKS LSLSPGK 107 IgG1 Fc domainEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT with Y407TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV mutationLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT (knob hole 1)LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKS LSLSPGK 108 IgG1 Fc domainEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT with T366WCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV mutationLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT (knob hole 2)LPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKS LSLSPGK 109 IgG1 Fc domainEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT with T366S,CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV L368A, andLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT Y407VLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP mutationsPVLDSDGSFFLVSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKS (knob hole 2) LSLSPGK 110Human IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVESCSVMHEALHNH YTQKSLSLSLGK 111Human IgG4 Fc PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ domainFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVESCSVMHEALHNHYTQKSLSLSLGK 112 Human IgG4ESKYGPPCPSCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVV Hinge + FcVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV domainLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVESCSVMHEALHNHYTQKSLSL SLGK 113 RepresentativeAESKYGPPCPPCPAPEAAGGPSVFLEPPKPKDTLMISRTPEVTCV Fc domainVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT sourced fromVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL DulaglutidePPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP (S228P, F234A,VLDSDGSFFLYSRLTVDKSRWQEGNVESCSVMHEALHNHYTQKSL L235A, L445P SLSLG-(EU) and K478del (Kabat) 114 Human IgG4ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA with mutationsLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPS K196Q, S228P,NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISR F296Y, E356K,TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTY R409K, andRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE H435RPQVYTLPPSQKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN mutations fromYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVESCSVMHEALHNR emicizumab YTQKSLSLSLGK115 Human IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAwith mutations LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSK196Q, S228P, NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRF296Y, R409K, TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTY and K439ERVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE mutations fromPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN emicizumabYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVESCSVMHEALHNH YTQESLSLSLGK 116Human IgG4 Fc PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ domain withFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY mutationsKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQKEMTKNQVS F296Y, E356K,LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK R409K, andLTVDKSRWQEGNVESCSVMHEALHNRYTQKSLSLSLGK H435R mutations from emicizumab117 Human IgG4 Fc PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQdomain with FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY mutationsKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS F296Y, R409K,LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK and K439ELTVDKSRWQEGNVESCSVMHEALHNHYTQESLSLSLGK mutations from emicizumab 118Human IgG4 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC Hinge + FcVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVS domain withVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ mutationsVYTLPPSQKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN S228P, F296Y,YKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALH E356K, R409K, NRYTQKSLSLSP--H435R, L445P, G446del (EU) and K478del(Kabat) mutations from emicizumab119 Human IgG4 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC Hinge + FcVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVS domain withVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ mutationsVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN S228P, F296Y,YKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEAL R409K, K439E, HNHYTQESLSLSP--L445P, G446del (EU)and K478del(Kabat) mutations from emicizumab 120Human IgG4 ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC Hinge + FcVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS domain withVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ mutationsVYVLPPSQEEMTKNQVSLLCLVKGFYPSDIAVEWESNGQPENN T350V, T366L,YLTWPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH K392L, T394W NHYTQKSLSLSLGK(zymeworks) 121 Human IgG4 ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCHinge + Fc VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS domain withVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ mutationsVYVYPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN T350V, L351Y,YKTTPPVLDSDGSFALVSRLTVDKSRWQEGNVFSCSVMHEALH F405A, Y407V NHYTQKSLSLSLGK(zymeworks) 122 Human IgG4 ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCHinge + Fc VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS domain withVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ mutation T366YVYTLPPSQEEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENN (knob hole 1)YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK 123Human IgG4 ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC Hinge + FcVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS domain withVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ mutation Y407TVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN (knob hole 1)YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK 124Human IgG4 ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC Hinge + FcVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS domain withVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ mutation T336WVYTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENN (knob hole 2)YETTPPVLDSDGSFFLYSRLTVDESRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK 125Human IgG4 ESKYGPPCPSCPAPEFLGGPSVFLFPPEPEDTLMISRTPEVTC Hinge + FcVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS domain withVLTVLHQDWLNGKEYECKVSNEGLPSSIEKTISKAKGQPREPQ mutationsVYTLPPSQEEMTKNQVSLSCAVEGFYPSDIAVEWESNGQPENN T366S, L368A,YETTPPVLDSDGSFFLVSRLTVDESRWQEGNVFSCSVMHEALH Y407V NHYTQKSLSLSLGK(knob hole 2) 126 Human IgG4 ESKYGPPCPPCPAPEFLGGPSVFLFPPEPEDTLMISRTPEVTCHinge + Fc VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS domain withVLTVLHQDWLNGKEYECKVSNEGLPSSIEKTISKAKGQPREPQ mutation S228PVYTLPPSQEEMTKNQVSLYCLVEGFYPSDIAVEWESNGQPENN (stability:YETTPPVLDSDGSFFLYSRLTVDESRWQEGNVFSCSVMHEALH reduced Fab NHYTQKSLSLSLGKarm exchange) 127 Human IgG4 ESKYGPPCPSCPAPEFEGGPSVFLFPPEPEDTLMISRTPEVTCHinge + Fc VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS domain withVLTVLHQDWLNGKEYECKVSNEGLPSSIEKTISKAKGQPREPQ mutation L235EVYTLPPSQEEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENN (decrease FcRYETTPPVLDSDGSFFLYSRLTVDESRWQEGNVFSCSVMHEALH binding) NHYTQKSLSLSLGK(Alegre et al 1992) 128 Human IgG4ESKYGPPCPSCPAPEAAGGPSVFLFPPEPEDTLMISRTPEVTC Hinge + FcVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS domain withVLTVLHQDWLNGKEYECKVSNEGLPSSIEKTISKAKGQPREPQ mutationVYTLPPSQEEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENN F234A, L235AYETTPPVLDSDGSFFLYSRLTVDESRWQEGNVFSCSVMHEALH (decrease FcR NHYTQKSLSLSLGKbinding) Xu et al Cell Immunol 2000 200(1):16-26) 129 Human IgG4ESKYGPPCPPCPAPEFEGGPSVFLFPPEPEDTLMISRTPEVTC Hinge + FcVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS domain withVLTVLHQDWLNGKEYECKVSNEGLPSSIEKTISKAKGQPREPQ mutationVYTLPPSQEEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENN S228P, L235EYETTPPVLDSDGSFFLYSRLTVDESRWQEGNVFSCSVMHEALH (decrease FcR NHYTQKSLSLSLGKbinding) Reddy et al., J Immunol 2000 164(4):1925- 33) 130 Human IgG4ESKYGPPCPSCPAPEFLGGPSVFLFPPEPEDTLMISRTPEVTC Hinge + FcVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVS domain withVLTVLHQDWLNGKEYECKVSNEGLPSSIEKTISKAKGQPREPQ mutation N297AVYTLPPSQEEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENN (affectsYETTPPVLDSDGSFFLYSRLTVDESRWQEGNVFSCSVMHEALH glycosylation,NHYTQKSLSLSLGK thus FcR binding) Borrok et al ACS Chem Biol 20127(9):1596-1602

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EQUIVALENTS

Those skilled in the art will recognize or be able to ascertain, usingno more than routine experimentation, many equivalents of the specificembodiments described herein. Such equivalents are intended to beencompassed by the following claims.

We claim:
 1. A composition comprising: (a) a first nucleic acid moleculecomprising a nucleotide sequence encoding a first polypeptide and asecond nucleic acid molecule comprising a nucleotide sequence encoding asecond polypeptide; or (b) a nucleic acid molecule comprising a firstnucleotide sequence encoding a first polypeptide and a second nucleotidesequence encoding a second polypeptide sequence; wherein the firstpolypeptide sequence and second polypeptide sequence are selected from:(i) a first polypeptide comprising the amino acid sequence of SEQ ID NO:40 and a second polypeptide comprising the amino acid sequence of SEQ IDNO: 43; (ii) a first polypeptide comprising the amino acid sequence ofSEQ ID NO: 41 and a second polypeptide comprising the amino acidsequence of SEQ ID NO: 42; (iii) a first polypeptide comprising theamino acid sequence of SEQ ID NO: 53 and a second polypeptide comprisingthe amino acid sequence of SEQ ID NO: 55; and (iv) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 57 and a secondpolypeptide comprising the amino acid sequence of SEQ ID NO: 59; (v) afirst polypeptide comprising the amino acid sequence of SEQ ID NO: 40,and a second polypeptide comprising the amino acid sequence selectedfrom SEQ ID NO: 43, SEQ ID NO: 55, and SEQ ID NO: 59; (vi) a firstpolypeptide comprising the amino acid sequence of SEQ ID NO: 43, and asecond polypeptide comprising the amino acid sequence selected from SEQID NO: 40, SEQ ID NO: 53, and SEQ ID NO: 57; (vii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 42, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:41, SEQ ID NO: 45, and SEQ ID NO: 47; (viii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 41, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:42, SEQ ID NO: 49, and SEQ ID NO: 51; (ix) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 53, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:43, SEQ ID NO: 55, and SEQ ID NO: 59; (x) a first polypeptide comprisingthe amino acid sequence of SEQ ID NO: 55, and a second polypeptidecomprising the amino acid sequence selected from SEQ ID NO: 40, SEQ IDNO: 53, and SEQ ID NO: 57; (xi) a first polypeptide comprising the aminoacid sequence of SEQ ID NO: 57, and a second polypeptide comprising theamino acid sequence selected from SEQ ID NO: 43, SEQ ID NO: 55, and SEQID NO: 59; (xii) a first polypeptide comprising the amino acid sequenceof SEQ ID NO: 59, and a second polypeptide comprising the amino acidsequence selected from SEQ ID NO: 40, SEQ ID NO: 53, and SEQ ID NO: 57;(xiii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 45, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 42, SEQ ID NO: 49, and SEQ ID NO: 51; (xiv) afirst polypeptide comprising the amino acid sequence of SEQ ID NO: 47,and a second polypeptide comprising the amino acid sequence selectedfrom SEQ ID NO: 42, SEQ ID NO: 49, and SEQ ID NO: 51; (xv) a firstpolypeptide comprising the amino acid sequence of SEQ ID NO: 49, and asecond polypeptide comprising the amino acid sequence selected from SEQID NO: 41, SEQ ID NO: 45, and SEQ ID NO: 47; (xvi) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 51, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:41, SEQ ID NO: 45, and SEQ ID NO: 47; (xvii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 61, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:67, SEQ ID NO: 75, and SEQ ID NO: 82; (xviii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 63, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:65, SEQ ID NO: 73, and SEQ ID NO: 81; (xix) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 65, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:63, SEQ ID NO: 71, and SEQ ID NO: 79; (xx) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 67, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:61, SEQ ID NO: 69, and SEQ ID NO: 77; (xxi) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 69, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:67, SEQ ID NO: 75, and SEQ ID NO: 82; (xxii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 71, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:65, SEQ ID NO: 73, and SEQ ID NO: 81; (xxiii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 73, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:63, SEQ ID NO: 71, and SEQ ID NO: 79; (xxiv) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 75, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:61, SEQ ID NO: 69, and SEQ ID NO: 77; (xxv) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 77, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:67, SEQ ID NO: 75, and SEQ ID NO: 82; (xxvi) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 79, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:65, SEQ ID NO: 73, and SEQ ID NO: 81; (xxvii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 81, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:63, SEQ ID NO: 71, and SEQ ID NO: 79; (xxviii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 82, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:61, SEQ ID NO: 69, and SEQ ID NO: 77; (xxix) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 84, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:89, SEQ ID NO: 97, and SEQ ID NO: 105; (xxx) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 85, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:87, SEQ ID NO: 95, and SEQ ID NO: 103; (xxxi) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 87, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:85, SEQ ID NO: 93, and SEQ ID NO: 101; (xxxii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 89, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:84, SEQ ID NO: 91, and SEQ ID NO: 99; (xxxiii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 91, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:89, SEQ ID NO: 97, and SEQ ID NO: 105; (xxxiv) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 93, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:87, SEQ ID NO: 95, and SEQ ID NO: 103; (xxxv) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 95, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:85, SEQ ID NO: 93, and SEQ ID NO: 101; (xxxvi) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 97, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:84, SEQ ID NO: 91, and SEQ ID NO: 99; (xxxvii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 99, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:89, SEQ ID NO: 97, and SEQ ID NO: 105; (xxxviii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 101, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:87, SEQ ID NO: 95, and SEQ ID NO: 103; (xxxix) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 103, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:85, SEQ ID NO: 93, and SEQ ID NO: 101; and (xl) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 105, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:84, SEQ ID NO: 91, and SEQ ID NO:
 99. 2. A recombinant expression vectorcomprising a nucleic acid encoding a first polypeptide according toclaim
 1. 3. A recombinant expression vector comprising a nucleic acidencoding a second polypeptide according to claim
 1. 4. A recombinantexpression vector comprising a nucleic acid encoding a first polypeptideand a second polypeptide according to claim
 1. 5. A host celltransformed with the recombinant expression vector of claim
 2. 6. A hostcell transformed with the recombinant expression vector of claim
 3. 7. Ahost cell transformed with the recombinant expression vector of claim 4.8. A host cell transformed with the recombinant expression vector ofclaim 2 and the recombinant expression vector of claim
 3. 9. A method ofproducing a heterodimer which binds type I interferons, the methodcomprising maintaining a cell according to claim 8 under conditionspermitting expression of the heterodimer.
 10. A method of producing aheterodimer which binds type I interferons, the method comprisingmaintaining a cell according to claim 7 under conditions permittingexpression of the heterodimer.
 11. The method of claim 10, wherein thetype I interferon is INFα.
 12. The method of claim 10, wherein the typeI interferon is INFβ.
 13. A method of decreasing interferon (IFN) geneexpression, inhibiting the activity of type I interferons, or treatingan autoimmune disease in a subject comprising administering to thesubject a heterodimer which binds type I interferons comprising a firstpolypeptide and a second polypeptide selected from: (i) a firstpolypeptide comprising the amino acid sequence of SEQ ID NO: 40 and asecond polypeptide comprising the amino acid sequence of SEQ ID NO: 43;(ii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 41 and a second polypeptide comprising the amino acid sequence ofSEQ ID NO: 42; (iii) a first polypeptide comprising the amino acidsequence of SEQ ID NO: 53 and a second polypeptide comprising the aminoacid sequence of SEQ ID NO: 55; and (iv) a first polypeptide comprisingthe amino acid sequence of SEQ ID NO: 57 and a second polypeptidecomprising the amino acid sequence of SEQ ID NO: 59; (v) a firstpolypeptide comprising the amino acid sequence of SEQ ID NO: 40, and asecond polypeptide comprising the amino acid sequence selected from SEQID NO: 43, SEQ ID NO: 55, and SEQ ID NO: 59; (vi) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 43, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:40, SEQ ID NO: 53, and SEQ ID NO: 57; (vii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 42, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:41, SEQ ID NO: 45, and SEQ ID NO: 47; (viii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 41, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:42, SEQ ID NO: 49, and SEQ ID NO: 51; (ix) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 53, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:43, SEQ ID NO: 55, and SEQ ID NO: 59; (x) a first polypeptide comprisingthe amino acid sequence of SEQ ID NO: 55, and a second polypeptidecomprising the amino acid sequence selected from SEQ ID NO: 40, SEQ IDNO: 53, and SEQ ID NO: 57; (xi) a first polypeptide comprising the aminoacid sequence of SEQ ID NO: 57, and a second polypeptide comprising theamino acid sequence selected from SEQ ID NO: 43, SEQ ID NO: 55, and SEQID NO: 59; (xii) a first polypeptide comprising the amino acid sequenceof SEQ ID NO: 59, and a second polypeptide comprising the amino acidsequence selected from SEQ ID NO: 40, SEQ ID NO: 53, and SEQ ID NO: 57;(xiii) a first polypeptide comprising the amino acid sequence of SEQ IDNO: 45, and a second polypeptide comprising the amino acid sequenceselected from SEQ ID NO: 42, SEQ ID NO: 49, and SEQ ID NO: 51; (xiv) afirst polypeptide comprising the amino acid sequence of SEQ ID NO: 47,and a second polypeptide comprising the amino acid sequence selectedfrom SEQ ID NO: 42, SEQ ID NO: 49, and SEQ ID NO: 51; (xv) a firstpolypeptide comprising the amino acid sequence of SEQ ID NO: 49, and asecond polypeptide comprising the amino acid sequence selected from SEQID NO: 41, SEQ ID NO: 45, and SEQ ID NO: 47; (xvi) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 51, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:41, SEQ ID NO: 45, and SEQ ID NO: 47; (xvii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 61, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:67, SEQ ID NO: 75, and SEQ ID NO: 82; (xviii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 63, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:65, SEQ ID NO: 73, and SEQ ID NO: 81; (xix) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 65, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:63, SEQ ID NO: 71, and SEQ ID NO: 79; (xx) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 67, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:61, SEQ ID NO: 69, and SEQ ID NO: 77; (xxi) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 69, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:67, SEQ ID NO: 75, and SEQ ID NO: 82; (xxii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 71, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:65, SEQ ID NO: 73, and SEQ ID NO: 81; (xxiii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 73, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:63, SEQ ID NO: 71, and SEQ ID NO: 79; (xxiv) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 75, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:61, SEQ ID NO: 69, and SEQ ID NO: 77; (xxv) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 77, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:67, SEQ ID NO: 75, and SEQ ID NO: 82; (xxvi) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 79, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:65, SEQ ID NO: 73, and SEQ ID NO: 81; (xxvii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 81, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:63, SEQ ID NO: 71, and SEQ ID NO: 79; (xxviii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 82, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:61, SEQ ID NO: 69, and SEQ ID NO: 77; (xxix) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 84, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:89, SEQ ID NO: 97, and SEQ ID NO: 105; (xxx) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 85, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:87, SEQ ID NO: 95, and SEQ ID NO: 103; (xxxi) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 87, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:85, SEQ ID NO: 93, and SEQ ID NO: 101; (xxxii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 89, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:84, SEQ ID NO: 91, and SEQ ID NO: 99; (xxxiii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 91, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:89, SEQ ID NO: 97, and SEQ ID NO: 105; (xxxiv) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 93, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:87, SEQ ID NO: 95, and SEQ ID NO: 103; (xxxv) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 95, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:85, SEQ ID NO: 93, and SEQ ID NO: 101; (xxxvi) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 97, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:84, SEQ ID NO: 91, and SEQ ID NO: 99; (xxxvii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 99, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:89, SEQ ID NO: 97, and SEQ ID NO: 105; (xxxviii) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 101, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:87, SEQ ID NO: 95, and SEQ ID NO: 103; (xxxix) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 103, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:85, SEQ ID NO: 93, and SEQ ID NO: 101; and (xl) a first polypeptidecomprising the amino acid sequence of SEQ ID NO: 105, and a secondpolypeptide comprising the amino acid sequence selected from SEQ ID NO:84, SEQ ID NO: 91, and SEQ ID NO:
 99. 14. The method of claim 13,wherein the type I interferon is INFα.
 15. The method of claim 13,wherein the type I interferon is INFβ.
 16. The method of claim 13,wherein the autoimmune disease is SLE or Sjogren's syndrome.